Dietary composition for adiposity reduction and cardiovascular health

Abstract

The present relates to a composition substantially comprising EAA and / or which does not substantially comprise NEAA. Such a composition is highly preferably for oral administration. Such compositions find use in the form of a solid food product, e.g. such as an animal feed, or in the form of a liquid, suspension or emulsion, e.g. in the form of a powder to be dispensed in a liquid for (human) consumption. Such a composition is provided to the subjects as part of their diet, or, their diet may consist of such a composition, taking into account the subject's individual dietary needs. Such use of the compositions, provides health benefits to the subjects being prescribed such diets, including weight loss while substantially retaining muscle mass.

Description

[0001] Title Dietary composition for adiposity reduction and cardiovascular health

[0002] Field of the invention

[0003] This invention relates to dietary compositions for use as supplements and / or food replacement in the treatment of various conditions. Such supplements and / or food replacement compositions are for use in the treatment of overweight or obesity and / or may have further health benefits, including on cardiovascular health and diabetes and related conditions, including in subjects at risk thereof.

[0004] Introduction

[0005] Obesity, cardiovascular disease, insulin resistance and / or type II diabetes, are diseases that are linked and believed to be mainly diseases of affluence in the western world to which diet and lifestyle are understood to be the main contributors. The contribution of diets to such diseases involves overnutrition, which causes alterations within white adipose tissue (WAT). Overnutrition is understood to lead to the induction of inflammatory signalling pathways, hypertrophy, and oxidative stress, which impair the ability of insulin to suppress lipolysis, leading to increased circulating free fatty acids (FFA). The resulting ectopic oversupply of lipids to the liver and muscle is understood to initiate a cascade of signalling events that compromise glucose control, and in the heart can lead to fibrotic tissue deposition and reduced myocardial blood flow, all of which manifest in reduced cardiometabolic function.

[0006] Dietary interventions, lifestyle changes, pharmaceutical interventions and even surgery are amongst the arsenal of treatment options available for subjects suffering from, or at risk from, such diseases. Dietary interventions represent a main avenue of research for potential treatment, or prevention, of such diseases, or, as part of a treatment plan. For example, consumption of high protein diets is known to significantly reduce adiposity, however long-term use of such diets has been implicated in a significant increase in risk of cardiovascular disease (Lagiou et al., BMJ, 2012). On the other hand, a low protein diet results in weight loss, but in the long term results in significant muscle loss as well with concomitant negative impact on body composition and health. The weight loss effects of such diets are understood to be the result of caloric restriction, and it is believed that proteins are essential to be included as part of the diet. Hence, there is a need in the art to provide further dietary interventions, which not only are aimed at reducing weight, but also allow for, in addition or separately, improvement of body composition and health thereby reducing risk of, or reverse or improve to a large extent, i.e. cardiovascular disease, insulin resistance and / or type II diabetes, and the like.

[0007] Summary of the invention

[0008] Non-essential amino acid (NEAA) depletion has been shown to have promise in cancer research, having an effect on slowing tumor growth and enhancing survival. In addition, supplementation with a defined single essential amino acid (EAA) has been shown to have some health benefits as well in some situations. In the present invention, because dietary proteins contain 9 EAA and NEAA amino acids, it was hypothesized that the amino acid profile may have an effect on the overall metabolic effect of protein in a diet, and hence may benefit the body composition and health of subjects in particular in subjects having obesity, cardiovascular disease, insulin resistance and / or type II diabetes. Highly advantageously, the present inventors found that a diet devoid of NEAA was shown to have a positive effect on weight loss, resulting i.a. in a significant loss of white adipose tissue. Highly surprisingly, the weight loss was not contributable to a lower caloric intake. In addition, increasing the EAA content of a diet highly advantageously resulted in even greater reduction of whole body and visceral fat mass. Moreover, highly surprisingly, the inventors found that such dietary intervention at the same time could provide for an overall improvement of cardiometabolic health, including one or more, or all, of a reduction of fasting blood glucose levels, a reduction of serum lipids, an improvement of glucose tolerance, and an improvement of cardiac output.

[0009] Hence, the present invention provides for means and methods with which to implement such dietary restrictions, i.e. provide additional EAA or provide EAA and avoid substantially NEAA. Natural proteins are comprised of both EAA and NEAA. In nature, proteins do not consist solely of EAA. Hence, a diet comprising food of e.g. plant and / or animal based origin and composed to comprise protein, will normally comprise a combination of both EAA and NEAA in the form of protein.

[0010] Hence, the present invention provides for a composition substantially comprising EAA and which does not substantially comprise NEAA. Such a composition is highly preferably for oral administration. As shown in the example section, such a composition may be in the form of a solid food product (e.g. such as an animal feed, as shown in the examples), or in the form of a liquid, suspension or emulsion (e.g. in the form of a powder to be dispensed in a liquid for human consumption, as outlined in the examples herein). Such a composition is provided to the subjects as part of their diet, or, their diet may be composed of such composition, taking into account the subject’s individual dietary needs. Without being bound by theory, such diets, which comprise the use of the compositions in accordance with the invention, therewith providing the health benefits to the subjects being prescribed such diets, as disclosed herein, such as shown in the examples.

[0011] Health benefits that may be associated with the use of compositions as defined herein, such as when part of a diet as outlined herein, include a reduction in body weight in subjects, e.g. human patients being overweight or obese, (e.g. as defined by having a BM I which exceeds 25) or at risk thereof. Without being bound by theory, health improvements that may be associated with the use of compositions as defined herein, such as when part of a diet as outlined herein, may be obtained in subjects, such as human patients, suffering from cardiomyopathy (such as diabetic cardiomyopathy), cardiometabolic disease, glucose intolerance, type II diabetes. With regard to the latter indication, it is understood that subjects suffering from obesity may in particular benefit, but it is understood that, without being bound by theory, subjects not necessarily suffering from obesity may benefit as well. Hence, the compositions for use as described herein may not necessarily be limited to the use in obese or overweight subjects and may find benefit in subjects which are not obese or overweight as well.

[0012] Figures

[0013] Figure 1. Anthropometries, body composition, and energy expenditure.

[0014] Changes in body weight compared to day 0 over the duration of the dietary intervention expressed as a percentage (A) and grams ( B), caloric intake (C, D), absolute change in body weight (E, F), hourly VO2 during dark and light cycles (G, H), VO2 (I, J), heat (K, L) and RER (M, N) measured during the 1 st and 2nd weeks respectively, histogram of animal body composition showing relative percentages of fat mass, lean mass and bone mass (O), micro-CT 3D reconstruction of fat and lean tissue distribution on animal skeletal frame (P), total body fat (Q), lean mass (R) and bone mass (S), eWAT representative images and normalized tissue weight (T), eWAT representative H&E histology stain images and adipocyte CSA (U), liver representative images and normalized tissue weight (V) and liver representative H&E histology stain images and hepatocyte CSA (W). Western blotting phosphorylated Akt was expressed as a ratio compared to total Akt content. Ponceau staining was used as a loading control. Values represent the mean ± SD; n = 6-36 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con, # p < 0.05 vs. 7% and + p < 0.05 vs. Pre. Con is control diet (white bars); 7% refers to 7% EAA diet (light grey bars); 18% refers to 18% EAA diet (dark grey bars). The same reference of Con, 7% and 18% is used in subsequent figures.

[0015] Figure 2. Cardiometabolic risk profiles and indices or cardiac function.

[0016] Serum FFA (A), TAG (B), GDF15 (C) and FGF21 (D), 4hr fasted blood glucose (E), incremental area under the curve calculated from 2hr IP glucose tolerance test (GTT) (F) fasted blood glucose measurements before and throughout the 15 minute period following insulin injection along with representative Western blot images and quantified ratios of phosphorylated / total AkT (G) and blood glucose levels throughout the 120 minute IP GTT (H), TA normalized tissue weight (I), RG fractional synthetic response (FSR) (J), liver representative H&E histology stain images and adiposity (K), eWAT representative picrosirius red stain images and fibrosis (L), heart representative images and normalized tissue weight (M) and FSR (N), left ventricle (LV) representative H&E histology stain images and CSA (O) and representative LV picrosirius red histology stain images and fibrosis (P), echocardiography representative B-mode and M-mode images (Q), PWT (R), comparative analysis of ESV and EDV (S), SV (T) and CO (U) taken from the LV. Values represent the mean ± SD; n = 5-20 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0017] Figure 3. Mitochondrial bioenergetics and mitochondrial H2O2 emission.

[0018] Titration of ADP in permeabilized fibre bundles from the RG and LV revealed typical Michaelis-Menton kinetics, which was used to determine the apparent ADP Km. Representative trace of mitochondrial respiration experiments (A), showing oxygen consumption (JO2) with complex I and II substrates (P = pyruvate, M = malate, D = ADP, G = glutamate, S = succinate, C = cytochrome C, RCR = respiratory control ratio; values normalized to fibre bundle dry weight for RG and LV, normalized to tissue wet weight for liver and brain and normalized to tissue wet weight and total cell count for eWAT) (C - RG; H - LV; M - liver; N - brain; O, P - eWAT), ADP-stimulated respiration showing the full Michaelis-Menton curve, and a rescaled x-axis to show the shift in respiration in each group (D - RG; I - LV), calculated apparent ADP Km (E - RG; J - LV), representative fluorometric trace (B) showing maximal succinate-stimulated H2O2 emission in the absence (F - RG; K - LV) and presence (G - RG; L - LV) of 100 pM ADP (values normalized to fibre bundle dry weight). Values represent the mean ± SD; n = 8-12 per group. Statistical analysis: Unpaired student t-test. * p < 0.05 vs. Con.

[0019] Figure 4. Transcriptomic analysis of epidydimal white adipose tissue following 4 days of 18% EAA consumption. Principle component analysis (PCA) showing gross expression patterns (A), volcano plot analysis of differential expressed genes (DEG) showing upregulated (red) and downregulated (blue) genes with > 1 fold change (B), cluster network analysis and gene-concept network diagram indicating downregulated DEGs corresponding to proteostasis and amino acid metabolism (C), KEGG enrichment analysis highlighting broad pathways pertaining to metabolism (D), Gene Ontology (GO) analysis highlighting top 10 downregulated metabolic processes (E) and heatmap showing sample-to-sample distances in key downregulated DEGs from GO enrichment analysis (F). Values represent the mean ± SD; n = 4-6 animals / group. p < 0.1.

[0020] Figure 5. T ranscriptomic analysis of the liver following 4 days of 18% EAA consumption.

[0021] Principle component analysis (PCA) showing gross expression patterns (A), volcano plot analysis of differential expressed genes (DEG) showing upregulated (red) and downregulated (blue) genes with > 1 fold change ( B), cluster network analysis and gene-concept network diagram indicating downregulated DEGs corresponding to fatty acid metabolism (C), KEGG enrichment analysis highlighting broad pathways pertaining to metabolism (D), Gene Ontology (GO) analysis highlighting top 10 downregulated metabolic processes (E) and heatmap showing sample-to-sample distance in key downregulated DEGs from GO enrichment analysis (F). Values represent the mean ± SD (P < 0.05); n = 4-6 animals / group. p < 0.1.

[0022] Figure 6. Analysis of conserved downregulated genes in epidydimal white adipose tissue and the liver following 4 days of 18% EAA consumption.

[0023] Venn diagram illustrating conserved overlapping downregulation of 102 genes between eWAT (adipose tissue; 854 genes) and liver tissues (Liver tissue; 334 genes) (A), KEGG enrichment analysis highlighting top 10 clusters of overlapping downregulated DEGs based on broad metabolic process (B), top 10 biological processes identified in GO enrichment analysis (C), comparative gene expression of all DEGs within the top 10 GO enrichment biological processes in eWAT (D) and liver (E) tissues, with white bars representing control and black 18% EAA consumption and a schematic demonstrating the involvement of conserved downregulated genes in fatty acid synthesis (highlighted in brown) (F). Values represent the mean ± SD; n = 4-6 animals / group. Statistical analysis: Unpaired students t-test. * p < 0.1 vs. Con.

[0024] Figure 7. Clinical translation of dietary NEAA depletion combined with EAA enrichment to a model of hyperphagic-induced obesity.

[0025] Comparative image of obese mouse vs. lean littermate (A), changes in body weight compared to day 0 over the duration of the dietary intervention expressed as a grams (B) and percentage (C), caloric intake (D, F), absolute change in body weight (E, G) during the 1 st and 2nd weeks, respectively, iWAT (H), heart (I) and liver (J) normalized tissue weight, 4hr fasted blood glucose (K), hourly VO2 during dark and light cycles (L), VO2 (M), heat (N) and RER (O), mitochondrial respiration in the presence of various saturating complex-l and complex-ll linked substrates (values normalized to fibre bundle dry) (P), ADP- stimulated respiration showing the full Michaelis-Menton curve, and a rescaled x-axis to show the shift in respiration in each group (Q), calculated apparent ADP Km (R), maximal succinate-stimulated H2O2 emission in the absence (S) and presence (T) of 100 pM ADP (values normalized to fibre bundle dry weight), echocardiography representative B-mode and M-mode images (U), PWT (V), comparative analysis of ESV and EDV (W), SV (X) and CO (Y) and comparative analysis with a linear regression of HR and SV (Z), LVP Max (Zi), LVP Min (Zii) and MAP (Ziii). Values represent the mean ± SD; n = 5-27 animals / group. Statistical analysis: Unpaired student t-test. * p < 0.05 vs. O Con.

[0026] Figure 8. Additional anthropometric and calorimetric data.

[0027] Changes in food consumption compared to day 0 over the duration of the dietary intervention (A, B), fecal energy content (C, D), VCO2 (E, H), heat (F, I) and ambulatory activity count (XAMB) (G, J). Values represent the mean ± SD; n = 7-36 animals / group. Statistical analysis: one way AN OVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0028] Figure 9. Micro-CT body composition data from week 1 .

[0029] Histogram of animal body composition showing relative percentages of fat mass (bottom area’s; light grey), lean mass (muscle mass, middle areas; grey) and bone mass (top areas; black) (A), micro-CT 3D reconstruction of fat and lean tissue distribution on animal skeletal frame (B), total body fat (C), lean mass (D) and bone mass (E). Values represent the mean ± SD; n = 7-36 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0030] Figure 10. Additional insulin-mediated phosphorylation targets in red gastrocnemius tissue.

[0031] Representative Western blot images and quantified values for phosphorylated / total eEF2 (A), AMPK (B), mTOR (C), ERK (D) and P70S6 (E). All Western blotting phosphorylated protein targets were expressed as a ratio compared to total protein content. Ponceau staining was used as a loading control. Values represent the mean ± SD; n = 7-12 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con, # p < 0.05 vs. 7% and + p < 0.05 vs. pre.

[0032] Figure 11. Additional eWAT and iWAT histology data. iWAT representative H&E histology stain images and adipocyte CSA (A), iWAT representative picrosirius red histology stain images and fibrosis (B), eWAT comparative analysis with a linear regression of cells per field of view and percent fibrosis (C - linear regression for each group; D - linear regression for all animals combined) and iWAT comparative analysis with a linear regression of cells per field of view and percent fibrosis (C - linear regression for each group; D - linear regression for all animals combined). Values represent the mean ± SD; n = 7-8 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con.

[0033] Figure 12. Additional tissue weights.

[0034] Normalized tissue weight for iWAT (A), soleus (B), kidney (C), lung (D) and pancreas (E) and tibia length (F). Values represent the mean ± SD; n = 7-8 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0035] Figure 13. Additional fractional synthetic response measurements. Fractional synthetic response measured in WG (A), liver (B), diaphragm (C), kidney (D), lung (E), pancreas (F), intestine (G), brain (H), skin (I) and bone (J), and plasma precursor measured across all tissues (K). Values represent the mean ± SD; n = 7-8 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0036] Figure 14. Additional mitochondrial bioenergetics and mitochondrial H2O2 emission data.

[0037] Mitochondrial respiration in the presence of various saturating complex-l and complex-ll linked substrates (values normalized to fibre bundle dry for WG; and normalized to tissue wet weight and total cell count for eWAT) (WG - A; iWAT - F, G), and in WG: ADP-stimulated respiration showing the full Michaelis-Menton curve and a rescaled x-axis to show the shift in respiration in each group (B), calculated apparent ADP Km (C), maximal succinate-stimulated H2O2 emission in the absence (D) and presence (E) of 100 pM ADP (values normalized to fibre bundle dry weight). Values represent the mean ± SD; n = 5-8 animals / group. Statistical analysis: unpaired student t-test. * p < 0.05 vs. Con.

[0038] Figure 15. Additional RNAseq data.

[0039] KEGG enrichment analysis using all DEF highlighting top 10 metabolic pathways in eWAT (A) and liver (B), PCA analysis showing gross expression patterns (C) and volcano plot analysis of DEGs showing upregulated and downregulated genes (red) in RG (D). Values represent the mean ± SD; n = 4-6 animals / group. Statistical analysis: unpaired student t-test. * p < 0.1 vs. Con.

[0040] Figure 16. Additional anthropometric and calorimetric data in obese animals.

[0041] Animal body weight on day 0 (A) and day 14 (B) of the dietary intervention and changes in food consumption compared to day 0 (C), VCO2 (D), heat (E) and ambulatory activity count (XAMB) (F), normalized tissue weight for pancreas (G), kidney (H) and lung (I) and tibia length (J). Values represent the mean ± SD; n = 5-12 animals / group. Statistical analysis: unpaired student t-test. * p < 0.05 vs. O Con.

[0042] Figure 17. Additional mitochondrial bioenergetics and mitochondrial H2O2 data in obese animals.

[0043] Mitochondrial respiration in the presence of various saturating complex-l and complex-ll linked substrates (values normalized to fibre bundle dry for RG; and normalized to tissue wet weight for eWAT and iWAT) (RG - A; eWAT - F; iWAT - H), ADP-stimulated respiration in RG showing the full Michaelis- Menton curve and a rescaled x-axis to show the shift in respiration in each group (B), calculated apparent ADP Km (C), maximal succinate-stimulated H2O2 emission in the absence (RG - D; eWAT - G) and presence (E) of 100 pM ADP (values normalized to fibre bundle dry weight for RG and to fibre wet weight for eWAT). Values represent the mean ± SD; n = 1 1-13 animals / group. Statistical analysis: unpaired student t-test. * p < 0.05 vs. O Con.

[0044] Figure 18. Additional echocardiography data in obese animals.

[0045] Echocardiograms were taken on day 0 and day 14 of the dietary intervention, and the following measurements were obtained: ESV (A), EDV (B), PWT (C), EF (D), FS (E), HR (F), SV (G) and CO (H). Values represent the mean ± SD; n = 17-20 animals / group. Statistical analysis: paired student t-test. p < 0.05 vs. Pre.

[0046] Figure 19. Additional echocardiography and invasive hemodynamics data in obese animals.

[0047] Indices of cardiac function including ESV (A), EDV (B), EF (C), FS (D) and HR (E) and invasive hemodynamics data including dP / dt max (F), dP / dt min (G), dP / dt @LVP40 (H), LVP @ dP / dt Max (I), LVEDP (J), SBP (K), DBP (L), Tau Weiss (M), Tau Glantz (N) and Tau Logistic (O). Values represent the mean ± SD; n = 5-20 animals / group. Statistical analysis: unpaired student t-test. * p < 0.05 vs. O Con.

[0048] Figure 20. Schematic overview of human study design.

[0049] Figure 21 : Sex differences were not apparent, as female mice displayed reductions in body weight (A) and fat mass (B). Male mice housed at thermoneutrality (28 degrees Celsius) displayed similar reductions in body weight (C) and fat mass (D). Values represent the mean ± SD; n = 36 animals / group for body weight, all other measurements n = 7-15 animals / group. * p < 0.05 vs. Con, # p < 0.05 vs. 7%.

[0050] Figure 22: Additional whole-body data in both sexes, thermoneutrality and matched for caloric intake. Absolute quantification of body fat (A), lean (B) and bone mass (C) in CT images. Hourly (D) and averaged (E) steady-state VO2 and energy expenditure (F) relative to body mass, and energy expenditure normalized to estimated lean mass (G) in male mice. In female mice, changes in glucose tolerance (H, I). Values represent the mean ± SD; n = 8-36 animals / group. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0051] Figure 23. Schematic overview of the downregulation of fatty acid synthesis which may increase lipid substrate availability for NEAA synthesis, compensating for the reduction in transcriptional programming involved in amino acid biosynthesis.

[0052] Figure 24. Serum metabolites. Serum free fatty acids (FFA: A), triacylglycerol (TAG: A), growth differentiation factor 15 (GDF15: B) and fibroblast growth factor 21 (FGF21 : B) were determined using ELISA, while untargeted metabolomics was utilized to examine amino acid metabolism (C-E). Volcano plot (C) depicting differentially affected metabolites, including the 7 metabolites displaying >1 -Iog2 fold change (annotated above). Pathway analysis (D) with individual amino acids changes highlighted in (E). Groups comparisons are depicted by shape (O and □), and the colour delineates the change. Values represent the mean ± SD; n = 5-7 animals / group. * p < 0.05 vs. Con.

[0053] Figure 25. Serum amino acid abundance. Serum essential (EAA) and non-essential amino acid (NEAA) abundance. Values represent the mean ± SD; n = 6 animals / group. * p < 0.05 vs. Con and # p < 0.05 vs. 7%.

[0054] Figure 26. Motif enrichment analysis in eWAT and liver. Figure 27. Proteomic analysis. Within eWAT, volcano plot of protein changes (A), heat map highlighting the 50 most affected proteins (B) and Gene Ontology (GO) analysis highlighting top 10 downregulated metabolic processes after consuming the 18% EAA diet for 14 days (C). Within the liver, volcano plot of protein (D), a heat map highlighting the 50 most affected proteins (E) and GO analysis highlighting top 10 downregulated metabolic processes after consuming the 18% EAA diet for 14 days (F). n = 4-6 animals / group.

[0055] Figure 28. Hourly VO2 during dark and light cycles (E), Respiratory exchange ratio (RER: A, B), rates of carbohydrate oxidation (C) and movement (D). Values represent the mean ± SD; n = 5-6 animals / group. Statistical analysis: unpaired student t-test. * p < 0.05 vs. O Con.

[0056] Detailed description

[0057] In one embodiment, the invention provides for a composition comprising a mixture of amino acids for use in the treatment of an overweight or obese subject, wherein the mixture of amino acids comprises essential amino acids and does not comprise non-essential amino acids and wherein the composition is administered orally. In another embodiment, the invention provides for a composition comprising a mixture of amino acids for use in the treatment of an overweight subject, wherein the mixture of amino acids comprises essential amino acids and does not comprise non-essential amino acids and wherein the composition is administered orally. In another embodiment, the invention provides for a composition comprising a mixture of amino acids for use in the treatment of an obese subject, wherein the mixture of amino acids comprises essential amino acids and does not comprise non-essential amino acids and wherein the composition is administered orally. It is understood that in accordance with the invention, the compositions may also be used in the treatment of subjects identified or diagnosed as being at risk of overweight or obesity.

[0058] It is understood that the treatment as contemplated herein relates to the treatment of subjects. Subjects that are contemplated for treatment in accordance with the invention include animals, such as domestic animals, including cats and dogs, and may include rodents and livestock as well. It is understood that mammals are highly preferred. The terms overweight or obese relate in general to the weight of subjects. Overweight or obesity can be classified through various measurements, such as of weight, length, and / or further physical aspects of the subjects body (e.g. waist circumference or the like, percentage body fat, etc. ). Being overweight or obese relates to conditions involving anexcess of bodily fat, and various means and methods are known in the art with which such an excess in body weight and / or bodily fat can be determined in subjects. Hence, various clinical classifications are widely available which can be used to identify and diagnose subjects as overweight or obese, or at risk thereof. In human subjects, measuring height and weight of a subject is used to calculate the parameter BMI, i.e. body mass index, which is a clinically widely accepted parameter for determining subjects being overweight or obese. BMI is calculated by dividing the weight in kilograms by the square of the height of the subject in meters (i.e. kg I m2). Hence, in one embodiment, the treatment of overweight and / or obese human patients comprises the treatment of human patients such classified having a BMI of at least 25. In general, adult human patients are classified as being overweight when having a BMI between 25 and 30, and classified as obese when having a BMI in excess of 30.

[0059] As said, in embodiments as described herein, the composition for use in the treatment of subjects in accordance with the invention comprises a mixture of amino acids comprising essential amino acids (EAA) which does not comprise non-essential amino acids (NEAA). It is understood that in such embodiments, because the composition is not to comprise non-essential amino acids, such compositions may be preferred not to comprise further proteins and / or peptides. This is because proteins and / or peptides, when originating from natural sources, e.g. animal, plant, microalgae, fungi, and edible insect origin, including fermented products thereof, as natural sources mainly comprise a substantial amount of non-essential amino acids. Of course, artifical proteins and / or artificial peptides may be selected and included in the composition, as long as these do not substantially comprise non- essential amino acids, such forms can be contemplated. Having amino acids provided as singular units (not as (poly)peptides) may be preferred, as this allows easy control of amounts of each essential amino acid. Hence, compositions in accordance with the invention preferably comprise amino acids in their free form. Other forms that may be contemplated include salts or esters thereof. Prodrug like- forms or other modified forms may be contemplated as well. It is understood amino acids as found in nature when having a chiral center are in the L-configuration, hence, amino acids in accordance with the invention are in the configuration as found in nature. As long as the form of amino acids selected allows for release of the amino acid after ingestion in the subjects body, i.e. is suitable for consumption, which allows the amino acid to be metabolised and / or assimilated by the subject’s body, such form of amino acid can be contemplated in accordance with the invention. Such amino acids may be derived from natural protein sources, e.g. through hydrolysis thereof. As long as the mixture of essential amino acids does not substantially comprise non-essential amino acids, such essential amino acids may be contemplated for use in the mixture of amino acids.

[0060] It is understood that the composition with regard to the amino acid composition part thereof is well defined. Other components that may be contemplated to be comprised therein, in accordance with the invention, may include other suitable compounds, e.g. minerals and vitamins, as well as carbohydrates and fats, dietary fiber, and the like, and flavourings. Combined, the composition may provide e.g. daily dietary requirements for the subject (e.g. in case the composition is the sole product consumed by a subject or in case it is as a supplement in which further useful dietary components may be comprised). The composition may also provide with further components that are useful to supplement a subject with. It is understood that adding further components furthermore may also aid in providing a composition which is well palatable for the subject. Providing e.g. a composition with EAA only dissolved or dispersed in water may have a less pleasant taste, and adding further components may improve taste and mouthfeel to make it more accommodating to (human) subject consumptions. There are no restrictions with regard to the further components to be included, and components that are commensurate with a healthy diet can be contemplated in order to provide and aide to health to the subject.

[0061] In another embodiment, provided is a composition for use in accordance with the invention, wherein the mixture of essential amino acids administered per day per subject, represents 5-30 %, or 5-10%, 6-9%, or about 7%; or 10-30%, 12 - 25%, 14 - 20%, 15-19 %, or about 17% of the total caloric intake. It is understood that recommended amounts of protein per day as part of a diet may guide the total amount of amino acids that is to be comprised in the composition combined with the administration schedule.

[0062] As shown in the examples herein, the amount of proteins as recommended in a diet, may be substantially replaced by an equivalent portion of EAA. This means that the amount of EAA that would normally be comprised in a recommended diet, is to be replaced with an equivalent amount. Hence, in one embodiment, the composition in accordance with the invention is administered to the subject to provide for an amount of EAA which is equivalent to the daily recommended amount of protein. In another embodiment, the composition in accordance with the invention is administered to the subject to provide for an amount of EAA which is equivalent to the amount of EAA in a protein as contained in a recommended diet. Such similar amounts are understood to be similar in terms of weight or caloric intake, e.g. in case of a daily intake of 100 grams of regular protein, comprising 40 grams of EAA, a daily intake of 40 grams of EAA can be taken. The amount of EAA representing EAA in their free form and their corresponding molecular weight.. Such equivalence may be in terms of weight. Such equivalent amount of EAA may be based on the amount, i.e. number of EAAs as comprised in regular protein. Of course, in such a scenario, the daily caloric intake represented by EAA is lower as compared with protein, and hence, the caloric intake may be compensated with fats and carbohydrates.

[0063] In further embodiments, the amount of protein in a recommended diet is replaced by the composition in accordance with the invention. In such embodiments, the daily recommended amount of protein, e.g. defined by caloric intake, is replaced with EAA. This means the amount of EAA administered to the subject will exceed the amount of EAA in protein as comprised in a normal diet (i.e. as in common diets in the art which are not in accordance with the invention) comprising regular protein sources. Hence, in another embodiment, the composition in accordance with the invention is administered to the subject to provide for an amount of EAA which is similar to or the same as the daily recommended amount of protein. In yet another embodiment, the composition in accordance with the invention is administered to the subject to provide for an amount of EAA which is similar to or the same to the amount of protein as contained in a recommended diet. Such similar amounts are understood to be similar in terms of weight or caloric intake, e.g. in case of a daily intake of 100 grams of regular protein, a daily intake of 100 grams of EAA can be taken. The amount of EAA representing EAA in their free form and their corresponding molecular weight. Such equivalence may be in terms of weight. Of course, the daily amount depends on the subject and accepted healthy diet selected. In these latter embodiments it is understood that the amount of EAA administered daily (e.g. as comprised in the composition daily administered) exceeds the amount of EAA as comprised in protein as part of a normal diet.

[0064] As shown in the examples herein, a diet with protein representing about 18% of the caloric intake, of which 7% was from EAA and 1 1 % from NEAA, has in one scheme its protein replaced with an equivalent amount of EAA representing the 7% EAA, and in another scheme the administered EAA amount represented 18% of the caloric intake. Having protein replaced by an equivalent amount of EAA (i.e. 18% protein vs. 7% EAA) provided for advantageous effects, which effects could be even more improved by exceeding the amount of EAA over 7% (e.g. by having 18% EAA instead of 18% protein). Hence, without being bound by theory, it is understood that having protein substantially replaced by a composition in accordance with the invention may provide benefit to subjects and / or having a relative excess of EAA in a diet in terms of daily consumption may provide even further benefit to subjects. Such benefits as observed in the the examples 1-4 herein are furthermore, without being bound by theory, believed to be well translatable to human subjects.

[0065] Hence, in yet another embodiment, provided is a composition for use in accordance with the invention, wherein the mixture of essential amino acids administered per day per human subject, represents an amount in the range of 5-30 % of the total caloric intake. In a further embodiment, the amount of EAA administered per day represents 5-10%, 6-9%, or about 7%, of the total caloric intake. In another further embodiment, the amount of EAA administered per day represents 10-30%, 12 - 25%, 14 - 20%, 15-19 %, or about 17% of the caloric intake per day for a human subject. A percentage of caloric intake may be regarded a useful measure to determine the amount of EAA to be administered, as that is commonly determined when advising or prescribing a diet to a subject, in particular human subjects, i.e. diets in the art may include commonly a recommended daily caloric intake.

[0066] In another embodiment, provided is a composition for use in accordance with the invention, wherein the mixture of essential amino acids administered per day per subject, represents an amount in the range of 5-30 % of the total caloric intake. In a further embodiment, the amount of EAA administered per day represents 5-10%, 6-9%, or about 7%, of the total caloric intake. In another further embodiment, the amount of EAA administered per day represents 10-30%, 12 - 25%, 14 - 20%, 15-19 %, or about 17% of the caloric intake per day for a subject. A percentage of caloric intake may be regarded a useful measure to determine the amount of EAA to be administered, as that is commonly determined and may be well controlled, e.g. in animal feed, when providing a diet to a subject, in particular animal subjects.

[0067] In accordance with the invention, in a further embodiment, the treatment comprises furthermore a diet prescribed to the subjects based on their individual needs. In yet another further embodiment, the diet prescribed to the subject further comprises fat and carbohydrates. As said, as understood in accordance with the invention, the composition may be composed such that it in itself provides for the diet. In such a scenario, the use of composition in accordance with the invention is to provide for the prescribed or recommended diet. Hence, the composition may be consistent with a recommended diet, in which the composition is the sole food or feed for the subject, which can obviously be well controlled in case of an animal food product or animal feed. Daily food intake obviously further including water consumption, which may comprise at least some minerals. For human subjects, such compositions and diets may also be contemplated.

[0068] Nevertheless, it may be preferred in subjects, in particular in human subjects, to have the composition in accordance with the invention as a supplement to be administered daily, combined with further food products. This is because of having meals is also important for social interaction and culture, providing benefit to subjects, in particular human subjects, as well. Hence, the prescribed diet to subjects, further to the composition of EAA administered, in one embodiment comprises mainly, and may consist mainly of, fats and carbohydrates as components from the three main food components, i.e. fats, carbohydrates, and protein, and in addition further useful nutrients, such that the protein component of the diet is substantially replaced by the composition in accordance with the invention as further outlined below. Combined, the diet prescribed, with the composition in accordance with the invention, provides for recommended caloric intake and nutritional value by the diet.

[0069] Hence, it is understood that a prescribed diet can consist of a daily intake of the composition in accordance with the invention, wherein the composition provides for the further nutritional value besides the EAA components, prescribed. Such a composition in accordance with the invention may be an animal feed or a food replacement product. When the composition in accordance with the invention is to be combined with a separately prescribed diet, it is understood that the prescribed diet combined with the composition in accordance with the invention, provides for the recommended caloric intake and nutritional value by the diet. Hence, in such a scenario, the composition in accordance with the invention is to be provided as a supplement to subjects.

[0070] In another embodiment, the caloric intake of a subject is determined by taking into account the physical activity of a subject. In a further embodiment, for human subjects, the caloric intake per day for a subject is calculated using a Harris-Benedict equation with a physical activity factor. In yet a further embodiment, the caloric intake per day is calculated with the following equation:

[0071] Caloric intake per day = Physical activity factor x [88.362 + (13.397 * body mass (kg)) + (4.799 x body height (cm)) - (5.677 x age (years))].

[0072] Hence, it is understood that caloric intake of a subject, in particular of human subjects, can be well determined, and hence, the amount of the composition in accordance with the invention can easily be determined as well. Of course, the above types of determinations may not be construed as limiting, and alternative and equally useful caloric intake determination may be applied.

[0073] As is understood from the above, the composition in accordance with the invention which may be used as a dietary supplement, in particular when being recommended or prescribed to a human subject, will include in its diet, furthermore mainly food which is low in protein. Hence, it may be understood that a human subject is to be prescribed a low protein diet, and provided with a composition in accordance with the invention, which combined, provides for the recommended diet to provide for the recommended total daily caloric intake and nutritional value. The prescribed diet may thus include regular food products which can be consumed by the subject, which regular food products are preferably selected to be low in protein content, as the composition in accordance with the invention is to provide for the EAA in place of regular protein.

[0074] Accordingly, in another embodiment, a composition for use in accordance with the invention is provided herein, wherein the prescribed diet consisting of a low protein diet, provides for an amount of protein with regard to total caloric daily intake of at most 5%, which is combined with the composition in accordance with the invention. It is understood that with regard to the low protein diet, the protein refers to the protein being comprised in common foods and does not refer to the EAA of the composition in accordance with the invention. More preferably, the total caloric daily intake of proteins in the prescribed diet can be selected to be lower, e.g. up to 4%, up to 3%, up to 2% or up to 1.5%. Alternatively, the regular foods as comprised in the diet provide for at most 20% relative to the caloric intake of EAA provided by the composition in accordance with the invention. As shown in the examples herein, suitable diets may comprise regular foods that provide for about 1 .4% of the daily caloric intake, whereas composition for EAA administration provide for about 7% or about 18% of the daily caloric intake.

[0075] Without being bound by theory, the advantageous effects as observed in the examples herein did not include any regular protein and provided only an EAA composition in accordance with the invention, but in order to achieve the advantageous effects, it may not be required to not provide any protein as part of the diet. Providing a diet without any NEAA does not mean that a subject will not have any NEAA, as at least protein breakdown by the subjects also provides for NEAA, the NEAA used by the subject will not all be required to be synthesized by the subject. Continuous breakdown protein provides a source of both EAA and NEAA, but of course this is limited as there is not a 100% recycling efficiency. Moreover, the effects observed were attributable to both a reduction in NEAA and / or increased EAA. In the examples as described herein, related to a human clinical trial, is provided for a low protein diet which may be suitable in accordance with the invention.

[0076] Hence, in another embodiment, a composition for use in accordance with the invention is provided, wherein the subject is prescribed a diet, which prescribed diet is a low protein diet comprising an amount of total protein per day representing at most 5% of the total caloric daily intake, which is combined with the composition in accordance with the invention. In a further embodiment, the composition for use in accordance with the invention is combined with a low protein diet, wherein the low protein diet comprises meals, wherein with each meal a portion of the composition in accordance with the invention is concomitantly administered. In another further embodiment, the prescribed low protein diet is for a human, wherein the meals comprise breakfast, lunch, dinner and a presleep snack. In yet further embodiments, the presleep snack comprise about 0.1 g of protein or less. It may also be contemplated to allow in the low-protein diet further snacks throughout the day, which comprise less than 0.5 g of protein per snack. It is understood that snacks may be allowed for in a prescribed diet, but diets can be contemplated to have a different schedule, e.g. having breakfast, lunch and dinner only, or, may have only lunch and dinner.

[0077] In any case, the low protein diet is to be combined with administration of the composition in accordance with the invention. This may be a single administration or this may be multiple administrations. It may be preferred to have the daily intake of the composition in accordance with the invention to be distributed equally between meals, i.e. at breakfast, lunch, and dinner, or at breakfast, lunch, dinner and evening snack. Hence, in a further embodiment, the daily intake of the composition is portioned between meals, for example, equally portioned between meals.

[0078] With regard to administration of the composition in accordance with the invention, e.g. in human subjects, it may furthermore be contemplated have the composition in accordance with the invention administered, e.g. with a prescribed low protein diet, in an intermittent fashion. This means that the composition in accordance with the invention and optional prescribed diet may be used for 7- 14 days, after which the subject follows a regular diet, for a defined period, after which the subject may use the composition in accordance with the invention for another 7-14 days, followed by another period with a regular diet, repeating the process until sufficient health benefit is obtained, or, repeating the process to maintain health benefits. It may be contemplated to implement the use of the composition in accordance with the invention (e.g. with low protein diet) in shorter intervals, e.g. every other day, every other two days, or the like. It may be contemplated to schedule the use of the composition in accordance with the invention (e.g. with low protein diet) for a defined number of days in a week, e.g. twice a week or thrice a week. It may be preferred however to use the composition in accordance with the invention (e.g. with low protein diet) for several days on end, e.g. 2, 3, 4, 5, 6 or 7 days on end, as such use may allow to obtain beneficial results more efficient. However, such use may be challenging from an adherence perspective point of view, in particular in human patients, hence, it may be contemplated to deviate from the preferred use of 7 days or more, of the composition in accordance with the invention (e.g. with low protein diet).

[0079] With regard to essential amino acids, it is preferred to provide for a composition in accordance with the invention which comprises all essential amino acids. With regard to non-essential amino acids, it is preferred to provide for a composition in accordance with the invention that does not comprise non-essential amino acids. It can be contemplated to not have all essential amino acids comprised in the composition, e.g. when prescribed with a low-protein diet that may already provide for some essential amino acids.

[0080] In an alternative embodiment, with regard to the composition in accordance with the invention, without being bound by theory, it is understood that the effects observed can be attributable to an increase, an excess in EAA in the diet. Hence, alternatively, any diet may be advantageously supplemented with an EAA composition in accordance with the invention, e.g. representing about 10% or more of the caloric daily intake, to therewith provide an excess of EAA, which may provide for advantageous effects as well. Accordingly, in such an alternative embodiment, it may not be required to have a low amount of NEAA in a diet. Hence, in such a scenario, the composition in accordance with the invention, either as a feed or food replacement or supplement, may provide for an excess of EAA to subjects as part of their daily diets to provide for advantageous effects such as shown in the example herein. Hence, the amount of EAA to be supplement in this alternative embodiment, may be in an amount which represents about 5%, 6%, 7%, 8%, 9%, 10%, or more of the recommended caloric intake. In the examples herein, having an excess of EAA representing about 1 1 % was shown to have effect.

[0081] In a preferred embodiment, the composition in accordance with the invention comprises the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In one embodiment, the composition in accordance with the invention which comprises a mixture of amino acids, which amino acids consist of the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In one embodiment, the composition in accordance with the invention which comprises amino acids, which amino acids consist of the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. It is understood that of course the composition may comprise further constituents besides the (mixture of) amino acids. In one embodiment, the composition in accordance with the invention comprises a mixture of amino acids which solely consists of the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine and which the mixture does not comprise non-essential amino acids alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine. In another embodiment, the composition in accordance with the invention comprises a mixture of amino acids which consists of the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine and optionally, other suitable constituents such as one or more of minerals, vitamins, carbohydrates, fats, dietary fiber, and flavourings, and further excipients. In another embodiment, the composition in accordance with the invention comprises a mixture of amino acids which consists of the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine and optionally, other suitable constituents such as one or more of minerals, vitamins, carbohydrates, fats, dietary fiber, and flavourings, and further excipients, wherein the composition does not comprise non-essential amino acids alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine, i.e. non-essential amino acids.

[0082] With regard to the amounts of each of the essential amino acids, amounts can be selected as determined of a suitable protein source or combination of protein sources as part of normal and healthy diets. In a further embodiment, the invention provides for a composition for use in accordance with the invention, wherein the composition comprises, in percent per weight relative to the weight total of amino acids in the composition, histidine 3-12 %, isoleucine 6-16 %, leucine 14-25 %, lysine 14-26 %, methionine 3-11 %, phenylalanine 4-16 %, threonine 5-17 %, tryptophan 0.5 - 6 %, and valine 9-20 %. In yet another further embodiment, the invention provides for a composition for use in accordance with the invention, wherein the composition comprises, in percent per weight relative to the weight total of amino acids in the composition, histidine 4-8 %, isoleucine 9-13 %, leucine 18-22 %, lysine 19-23 %, methionine 4-8 %, phenylalanine 8-12 %, threonine 8-12 %, tryptophan 1-5 %, and valine 12-16 %. It is understood that these percentages are calculated based on these amino acids in their free form as listed below.

[0083] Table 1 Essential Amino Acids

[0084] In yet a further embodiment, the composition for use in accordance with invention as described herein comprises, in percent per weight relative to the weight total of amino acids in the composition, Histidine 5-7 %, Isoleucine 10-12%, Leucine 19-21 %, Lysine 20-22%, methionine 5-7%, Phenylalanine 9-11 %, Threonine 9-11 %, tryptophan 2-4%, and valine 13-15%. In yet another further embodiment, the composition for use in accordance with the invention comprises, in percent per weight relative to the weight total of amino acids in the composition, Histidine 5.8 %, Isoleucine 10.7%, Leucine 20.3%, Lysine 21.2%, Methionine 5.8%, Phenylalanine 9.8%, Threonine 9.4%, tryptophan 2.7%, and valine 14.3%. The latter defined composition of EAA is derived from the composition of milk protein, which is the composition which was used in the examples throughout as milk is considered a suitable source of protein in a healthy diet. Of course, other sources of protein may be contemplated to use as a reference for the amounts of the EAA in the composition in accordance with the invention, and may equally provide for the advantageous effects as described herein. Nevertheless, advantageous effects were obtained with such a defined composition and hence, such a composition may be preferred.

[0085] As said, the composition is preferably not to comprise non-essential amino acids. Hence, the composition in accordance with the invention preferably does not comprise the non-essential amino acids alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine.

[0086] As said, some observations have been made with the use of EAA, or depletion of a NEAA, in subjects with cancer. It is observed that cancer is a very different disease state from the diseases or health indices on which the present invention is to have an effect, i.e. overweight or obese subjects, and providing i.a. a significant loss of white adipose tissue, and / or provide for overall improvement of cardiometabolic health, including one or more, or all, of a reduction of fasting blood glucose levels, a reduction of serum lipids, an improvement of glucose tolerance, an improvement of cardiac output. These advantageous health effects may be obtained, e.g. in human patients diagnosed with conditions which may benefit from such improvements, by administering orally to such patients, a composition in accordance with the invention. Without being bound by theory, the composition being used either as a supplement, e.g. with a low protein diet, or, the composition being used as a food substitute wherein protein has been substantially been replaced with the EAA. The composition in accordance with the invention is preferably not for use in subjects suffering from cancer, e.g. is not for use in the treatment of cancer in such subjects. Hence, in a further embodiment, the composition in accordance with the invention, is not for use in subjects suffering from cancer, or, is for uses wherein the subjects are not suffering from cancer. In one embodiment, the composition is for use in human subjects, wherein the age of the subject is at least 8, 12, 16, or 18 years. In another embodiment, the composition is for use in human subjects, wherein the age of the subject is at most 35, 45, 55, or 65 years. In a further embodiment, the age of the human subjects is selected from the range of 8 - 35 years, 12 - 35 years, 16 - 35 years, 18 - 35 years, 8 - 45 years, 12 - 45 years, 16 - 45 years, 18 - 45 years, 8 - 55 years, 12 - 55 years, 16 - 55 years, 18 - 55 years, 8 - 65 years, 12 - 65 years, 16 - 65 years, or 18 - 65 years, In one embodiment, the age of the human subjects is selected from the range of 12 - 65 years or 16 - 65 years. In yet a another embodiment, the composition in accordance with the invention finds use in medical treatments of human subject, wherein the subject is female or male.

[0087] In one embodiment, the composition in accordance with the invention, is for use in the treatment of subjects, wherein in the treatment, subjects have a reduction in body weight. In one embodiment, the composition is for use in the treatment of obesity of in a subject, or for use in the treatment of a subject at risk of obesity. In another embodiment, the composition is for use in the treatment of obesity of in a subject, or for use in the treatment of a subject at risk of obesity, wherein the subject is prescribed a low protein diet. It is understood that preferably in medical treatments of subjects diagnosed with the diseases or at being at risk of disease as outlined herein, the composition in accordance with the invention comprises a mixture of amino acids, wherein the mixture of amino acids consists of the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine (i.e. the mixture does not comprise non-essential amino acids alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine), wherein the composition is administered orally, and the subject is prescribed a low protein diet. In another embodiment, in the treatment, subjects muscle mass is substantially maintained. In another embodiment, in the treatment, skeletal muscle protein synthesis rates are substantially maintained. In yet another embodiment, in the treatment, subjects have an increase in whole body fat oxidation. As observed in the examples herein, the use of the composition in the treatment of subjects, e.g. overweight or obese subjects, resulted in a reduction in whole body fat, in particular of visceral fat and white adipose tissue, while the muscle mass was largely maintained. In one embodiment, in the treatment, subjects have a reduction in visceral fat. In another embodiment, in the treatment, subjects have a reduction in adiposity. In a further embodiment, the composition in accordance with the invention, is for use in the treatment of subjects, wherein in the treatment, subjects have a reduction in (epidymal) white adipose tissue and / or a reduction in whole body fat. As shown in the examples, it was in particular observed that lipid synthesis in liver and white adipose was significantly reduced. Hence, the composition in accordance with the invention, is for use in the treatment of subjects, wherein in the treatment, subjects have a reduced hepatic adiposity or reduced hepatic lipid accumulation and / or subjects have a reduction in lipid synthesis within white adipose tissue and liver.

[0088] It is understood that with regard to the above mentioned effects which can be obtained in subjects with the compositions in accordance with the invention, as outlined in the examples herein, and as known in the art, such effects can be well determined by the skilled person. The extent of the effect as is well understood by the skilled person depends on the duration of the intervention and / or the condition of the subject.

[0089] In addition to the above improvements with regard to obese and overweight subjects, further improvements were observed, as shown in the examples herein, that independently or concomitantly improved in subjects provided with a composition in accordance with the invention. Hence, attaining such improvements in subjects provides for further uses of the composition in accordance with the invention which may benefit subjects, such as human subjects, being diagnosed with conditions which may benefit from such improvements. Hence, the compositions in accordance with the invention are of use in subjects diagnosed with, suspected of having, or at risk of, certain indications which may benefit from the health improvements as observed with the use of composition in accordance with the invention as outlined herein.

[0090] Accordingly, in one embodiment, the composition in accordance with the invention is for use in subjects diagnosed with or suspected of having diabetic cardiomyopathy. In another embodiment, the composition is for use in the treatment of diabetic cardiomyopathy. In a further embodiment, in the treatment of subjects with diabetic cardiomyopathy, diabetic cardiomyopathy is reversed. In another embodiment, the composition is for use in subjects wherein in the treatment indices of heart function are not negatively affected. The latter makes this treatment for use in patients at risk of or having heart disease. Moreover, it was observed that cardiometabolic health was improved. Hence, in another embodiment, the use of the composition in accordance with the invention as outlined herein, comprises an improvement of cardiometabolic health. In a further embodiment, said improvement of cardiometabolic health comprises one or more of: reduced fasting blood glucose levels, reducing serum lipids, improving glucose tolerance, improving cardiac output. In a further embodiment, the composition according the invention is for use in subjects, wherein the subjects do not suffer from obesity or being overweight, as it may be understood that these health benefits are of use not solely to such subjects.

[0091] As shown in the examples herein, when the composition is used in the treatment of subjects, this has an effect on gene expression, i.e. some genes were modulated (increased or decreased). Such effects on gene expression may be of use to monitor e.g. in subjects undergoing the treatments as outlined herein, e.g. to assess efficacy of treatment. Such monitoring may involve analysing suitable samples obtained from subjects, e.g. such as from samples as outlined in example 5 and shown Figure 20. Highly preferably, such samples are obtained with non-invasive or minor invasive procedures and any suitable analysis may be employed (i.a. analysis of protein, RNA, or metabolites or catabolites). Hence, genes as listed in the examples herein as being modulated, i.e. being increased or decreased, may be advantageously monitored in the treatment of subjects with the compositions in accordance with the invention, e.g. to monitor the effect of the treatment. Hence, in one embodiment, the composition in accordance with the invention when used in a treatment of a subject decreases gene expression involved in lipid storage, preferably within eWAT and liver. In another embodiment, the composition in accordance with the invention, when used in a treatment of a subject, decreases gene expression, which includes genes involved in metabolic processes, such as carbon, amino and fatty acid metabolism. In another embodiment, the composition in accordance with the invention when used in a treatment of a subject, induces a decrease in gene expression, which includes genes involved in fatty acid metabolism and / or synthesis, including one or more of Acly, Fasn, Acaca and Elovl6l. In yet another embodiment, the composition in accordance with the invention when used in a treatment of a subject, induces modulation in genes associated with one or more of Kruppel-like factor-5 (KLF5), Specificity protein 9 (SP9), and KLF12

[0092] EMBODIMENTS

[0093] 1 . A composition comprising a mixture of amino acids for use in the treatment of an obese or overweight subject, wherein the mixture of amino acids comprises essential amino acids and does not comprise non-essential amino acids and wherein the composition is administered orally.

[0094] 2. The composition for use in accordance with embodiment 1 , wherein the mixture of essential amino acids administered per day per human subject, represents about 5-30, or about 15-19, or about 17% of the total caloric intake.

[0095] 3. The composition for use in accordance with embodiment 1 or embodiment 2, wherein the treatment comprises furthermore a diet prescribed to the subjects based on their individual needs. The composition for use in accordance with embodiment 3, wherein the diet comprises fat and carbohydrates. The composition for use in accordance with embodiment 4, wherein the caloric intake per day for a subject is calculated using a Harris-Benedict equation with a physical activity factor. The composition for use in accordance with embodiment 5, wherein the caloric intake per day is calculated with the following equation:

[0096] Caloric intake per day = Physical activity facto (4.799 x body height (cm)) - (5.677 x age (years)) The composition for use in accordance with any of embodiments 3-6, wherein the prescribed diet is a low protein diet comprising an amount of total protein per day representing at most 5% of the total caloric daily intake. The composition for use in accordance with any of embodiments 3-7, wherein the low protein diet comprises meals, wherein with each meal a portion of the composition is concomitantly administered. The composition for use in accordance with embodiment 8, wherein the meals comprise breakfast, lunch, dinner and a pre-sleep snack. The composition for use in accordance with any of embodiments 8-9, wherein the pre-sleep snack comprise about 0.1 g of protein or less. The composition for use in accordance with any of embodiments 8-10, wherein the low-protein diet comprises further snacks throughout the day, which comprise less than 0.5 g of protein per snack. The composition for use in accordance with any of embodiments 8-11 , wherein the daily intake of the composition is equally portioned between meals. The composition for use in accordance with any of embodiments 1-12, wherein the essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine . The composition for use in accordance with any of embodiments 1-13, wherein the composition comprises, in percent per weight relative to the weight total of amino acids in the composition, histidine 4-8 %, isoleucine 9-13 %, leucine 18-22 %, lysine 19-23 %, methionine 4-8 %, phenylalanine 8-12 %, threonine 8-12 %, tryptophan 1-5 %, and valine 12-16 %. 15. The composition for use in accordance with any of embodiments 1-14, wherein the composition comprises, in percent per weight relative to the weight total of amino acids in the composition, Histidine 5-7 %, Isoleucine 10-12%, Leucine 19-21 %, Lysine 20-22%, methionine 5-7%, Phenylalanine 9-11 %, Threonine 9-11 %, tryptophan 2-4%, and valine 13-15%.

[0097] 16. The composition for use in accordance with any of embodiments 1-14, wherein the composition comprises, in percent per weight relative to the weight total of amino acids in the composition, Histidine 5.8 %, Isoleucine 10.7%, Leucine 20.3%, Lysine 21.2%, Methionine 5.8%, Phenylalanine 9.8%, Threonine 9.4%, tryptophan 2.7%, and valine 14.3%.

[0098] 17. The composition according to any of embodiments 1-16, wherein the non-essential amino acids are alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine.

[0099] 18. The composition according to any of embodiments 1-17, wherein the subjects are not suffering from cancer.

[0100] 19. The composition according to any of embodiments 1-18, wherein in the treatment, subjects have a reduction in body weight.

[0101] 20. The composition according to any of embodiments 1-19, wherein in the treatment, subjects muscle mass is substantially maintained.

[0102] 21. The composition according to any of embodiments 1-20, wherein in the treatment, subjects have an increase in whole body fat oxidation.

[0103] 22. The composition according to any of embodiments 1-21 , wherein in the treatment, subjects have a reduction in visceral fat.

[0104] 23. The composition according to any of embodiments 1-22, wherein in the treatment, subjects have a reduction in adiposity.

[0105] 24. The composition according to any of embodiments 1-23, wherein in the treatment, subjects have a reduction in (epidymal) white adipose tissue.

[0106] 25. The composition according to any of embodiments 1-24, wherein in the treatment, subjects have a reduction in whole body fat.

[0107] 26. The composition according to any of embodiments 1-25, wherein in the treatment, subjects have a reduced hepatic adiposity or reduced hepatic lipid accumulation. 27. The composition according to any of embodiments 1-26, wherein in the treatment, subjects have a reduction in lipid synthesis within white adipose tissue and liver.

[0108] 28. The composition according to any of embodiments 1-27, wherein the subjects have a diabetic cardiomyopathy.

[0109] 29. The composition according to any of embodiment 28, wherein in the treatment, diabetic cardiomyopathy is reversed.

[0110] 30. The composition according to any of embodiments 1-29, wherein in the treatment indices of heart function are not negatively affected.

[0111] 31. The composition according to any of embodiments 1-30, wherein in the treatment, cardiometabolic health is improved.

[0112] 32. The composition according to any of embodiments 1-31 , wherein in the treatment, the improvement of cardiometabolic health comprises one or more of: reduced fasting blood glucose levels, reducing serum lipids, improving glucose tolerance, improving cardiac output .

[0113] 33. The composition according to any of embodiments 1 -32, wherein in the treatment, skeletal muscle protein synthesis rates are substantially maintained.

[0114] 34. The composition according to any of embodiments 1 -33, wherein the subjects do not suffer from obesity or being overweight.

[0115] 35. The composition according to any of the embodiments 1-34, wherein the use of the composition in a subject decreases gene expression involved in lipid storage, preferably within eWAT and liver.

[0116] 36. The composition according to claim 35, wherein the decrease in gene expression includes genes involved in metabolic processes, such as carbon, amino and fatty acid metabolism.

[0117] 37. The composition according to claim 35 or 36, wherein the decrease in gene expression includes genes involved in fatty acid metabolism and / or synthesis, including Acly, Fasn, Acaca and Elovl6.

[0118] 38. The composition according to any of the embodiments 1-36, wherein the use of the composition in a subject, induces modulation in genes associated with one or more of Kruppel-like factor-5 (KLF5), Specificity protein 9 (SP9), and KLF12. 39. The composition according to any of claims 1 -37, wherein the age of the subject which is treated, is between 12 and 65 years, preferably between 18 and 65 years.

[0119] 40. The composition according to any of claims 1 -38, wherein the subject which is treated is a human subject and wherein the human subject is male or female.

[0120] EXAMPLES

[0121] MATERIALS AND METHODS FOR EXPERIMENTS 1-4

[0122] Experimental overview

[0123] Lean animals: Male C57BI / 6N mice were bred in-house at the University of Guelph (22°C) on a 12: 12 light dark cycle, and provided 24 h access to food and water ad libitum. At 18-22 weeks of age, mice were randomly assigned to receive a control diet (18% energy from protein, A11112201 B), a modified control diet void of non-essential amino acids (7% energy from essential amino acids: EAA, A20102001 ), or a nitrogen-matched modified control diet void of non-essential amino acids (18% energy from EAAs, A20102002) for a period of 2 weeks (n = 40-41 per group; see table 1 for detailed breakdown of each diet). All diets were purchased from Research Diets (New Brunswick, NJ, USA). Animals within each group were utilized for calorimetric analysis (n = 12-15 per group), glucose tolerance testing (n = 12-13 per group), whole-body micro-CT analysis and 3D rendering (n = 10 per group), fecal energy analysis (n = 7-8 per group) and echocardiography analysis (n = 14-15 per group). Tissue samples were taken for histological analysis (n = 7-8 per group), Western blotting (n = 7-9 per group), tissue protein synthesis rates (n = 7-8 per group), high resolution respirometry (n = 7-8 per group), mitochondrial reactive oxygen species emission (ROS: n = 7-8 per group) and proteomic analysis (n = 6 per group). A separate group of mice were randomly selected and fed identical diets for 4 days, after which whole body micro-CT analysis and 3D rendering (n = 3-4 per group), calorimetric measurements (n = 6-8 per group) and tissue samples for transcriptomic analysis (n = 6 per group) were taken.

[0124] Table 2: Composition of the experimental diets.

[0125] Obese animals: In separate experiments, in-house spontaneously obese male and female mice between the ages of 3 months to 1 year were randomly assigned fed identical diets for a period of 2 weeks (Con - n = 22; 18% EAA - n = 27). All lean littermates (n = 18) were supplemented the control diet. Animals within each group were utilized for calorimetric analysis (n = 4-7 per group), glucose tolerance testing (n = 9-13 per group), echocardiography analysis (n = 13-19 per group), invasive hemodynamics (n = 4-5 per group), high resolution respirometry (n = 9-13 per group) and mitochondrial ROS emission (n = 9-13 per group). During each round of experimentation, animal body weight and food intake were measured every 2 days. A 48 h washout period was implemented in groups of animals where multiple experiments were to be conducted. Detailed descriptions of each experimental and surgical protocol can be found in the sections below. All experiments were approved by the Animal Care Committee at the University of Guelph (AUP - 4241 ) and were consistent with the ARRIVE guidelines and regulations for reporting experiments involving animals. All efforts were made to minimize suffering of animals.

[0126] Metabolic characterization

[0127] CLAMS: Animal movement, heat production, resting oxygen consumption (Vo2) and carbon dioxide production (Vco2) were monitored in metabolic caging (Columbus Instruments, Columbus, OH, USA). Vo2 and Vco2 were used to calculate respiratory exchange ratio and energy expenditure, as previously reported (41 ).

[0128] Glucose tolerance testing: Following a 4 h fast, a glucose tolerance test was performed Briefly, the tip of the tail (<1 mm) was cut with scissors following the fast period, and fasting blood glucose measurements were taken using a glucose strip (Freestyle Lite, Abbott Diabetes Care, Alameda, CA, USA). Blood glucose levels were then monitored at the 0, 15, 30, 45, 60, 90, and 120 min timepoints following intraperitoneal glucose injection (2 g glucose / kg animal body weight), and the area under the curve was calculated as the product of above baseline values for each mouse (41 ). All animals were fasted from 08:00-12:00 h in order to eliminate time of day as a confounding variable.

[0129] Insulin-mediated signalling: Following a 4 h fast, mice were anaesthetized using a 2% isoflurane gas mixture. A red gastrocnemius muscle was surgically removed and snap frozen in liquid nitrogen prior to an I.P injection of insulin (Novorapid: 1 U / kg animal body weight). 15 min postinjection, the contralateral red gastrocnemius muscle was surgically removed and snap frozen in liquid nitrogen. Blood glucose levels were monitored at the 0, 10 and 15 min timepoints. Tissues were used for subsequent Western blotting analysis (see below for more detailed protocol). Procedures were only performed following confirmation of anaesthesia depth through palpebral reflex, whisker movement, or leg retraction after toe pinch.

[0130] CT Scanning

[0131] Mice were imaged using a micro-computed tomography (CT) scanner (SkyScan 1278, Bruker, Billerica, MA, USA) over 360° of gantry rotation at 0.25° angle increments, with X-ray tube potential of 50kV, tube current of 1005uA, exposure time of 50 ms and 12 frames per view angle averaged. Briefly, 2D projection images were transformed into 3D volume batches with 15% beam hardening, a ring artifact correction of 2, and gaussian smoothing of 2 using the NRecon program (Bruker, Billerica, MA, USA). Next, 3D volume batches corresponding to each mouse were reconstructed and analyzed using the CTan program (Bruker, Billerica, MA, USA). Following analysis, reconstructed images were visualized in the CTvol program (Bruker, Billerica, MA, USA), and rep 3D models were generated. For a detailed description of the protocol developed to complete this analysis, see Appendix 1.

[0132] Fecal energy analysis

[0133] Fecal pellets (~2 g wet weight) were collected from mice, and energy content was analyzed via bomb calorimetry. Briefly, fecal samples were mixed with 3.5><dH2O (w / v) and pulse-homogenized for a period of -5 min into a homogenous slurry using a Qiagen TissueRuptor (Qiagen Sciences, Maryland, USA). Fecal slurries were then freeze-dried for 24 h and subsequently pelleted. Fecal pellet energy content was measured using a C6000 bomb calorimeter (IKA Works Inc, North Carolina, USA) and gross energy was reported as Kcal / g.

[0134] Echocardiography analysis

[0135] In vivo assessment of cardiac structure and morphology were examined in non-fasted mice between 6 pm and 10 pm using the Vevo2100 microimaging system (VisualSonics, Toronto, ON, Canada). Mice were anaesthetized using an isoflurane / oxygen (5%:95%) gas mixture in a sealed chamber, and were transferred to a heated handling table, where anaesthesia was maintained with integrated isoflurane / oxygen ( 1 -2%: 98-99%) below the level of pedal reflex. Body temperature was maintained at 37 °C. High resolution images were taken within 20 min of MS550D ultrasound transducer induction (set to 40 MHz) across the left parasternal long axis. All measurements were obtained using the Cardiac Package (VisualSonics, Toronto, ON, Canada).

[0136] LV traces: B-mode (2D) images were acquired so in a manner such that apex to outflow tract within the LV chamber could be clearly visualized. M-mode (1 D) images were taken at the mid papi I lary level, and the LV-trace function was utilized to analyze 3-5 cardiac cycles per M-mode image.

[0137] Pulmonary flow traces: Pulsed-wave Doppler echocardiography was utilized. The transducer was positioned 1-2 mm superior to the LV imaging plane, and measurements were taken from the pulmonary trunk in a location immediately distal to the semilunar valves. Pulmonary flow was calculated as the product of the velocity-time integral (VTI) and vessel area (2rrr2). Right ventricular CO was the product of VTI X animal heart rate (HR). Measurements for VTI were obtained at the semilunar valve leaflets, and measurements for vessel diameter were taken immediately distal from this location. A beam angle of <45° was used, along with Doppler gain between 32-35 dB at a frequency of 32 MHz to minimize colour Doppler variability.

[0138] Invasive hemodynamics

[0139] Mice were anesthetized as described above below the level of pedal reflex. Following this, an incision was made along the anterior region of the neck, and a 1 ,2F pressure catheter (FTS-1211 B-0018; Scisense Inc., London, ON, Canada) was carefully inserted into the left ventricle via the right common carotid artery. Hemodynamic signals were digitized at a sampling rate of 2kHz and were analyzed using Spike2 v10 software (CED Spike2, Cambridge, UK).

[0140] Tissue collection

[0141] Non-fasted mice were anaesthetized via I.P injection of sodium pentobarbital (60 mg kg-1 ). Diverse tissues were removed, and prepared for the following procedures: Fixed for histological analysis, immediately sectioned for the assessment of mitochondrial respiration and ROS emission, and / or snap frozen in liquid nitrogen for Western blotting, tissue protein synthesis, transcriptomic or proteomic analysis, as described below.

[0142] Histological analysis Cell cross-sectional area (CSA) and liver adiposity: Epididymal (eWAT) and inguinal (iWAT) white adipose, LV and liver tissues were fixed, embedded and stained with hematoxylin and eosin (H&E. Images were taken using an Olympus FSX100 light microscope at a magnification of 40x with a 3X3 stack for adipocyte and liver slides, and with a 5X5 stack for LV slides. Analysis was completed using Imaged software (National Institute of Health, Bethesda, MD, USA). Briefly, CSA was calculated as the average of all cells within the field of view. Liver adiposity was calculated via thresholding analysis of 4 different capillary-free regions within the field of view. Black regions were defined as hepatocytes, and white regions were defined as adipose within the hepatocytes. Adiposity was expressed as a percentage of total tissue area.

[0143] Tissue fibrosis: Epididymal (eWAT) and inguinal (iWAT) white adipose and LV tissues were removed, embedded and stained with picrosirius red, as previously described (Petrick et al, 2023). Images were taken using an Olympus FSX100 light microscope at a magnification of 40x with a 3X3 stack for adipocyte slides, and with a 5X5 stack for LV slides. Under a light microscope, picrosirius staining reveals cytoplasm and cardiac fibres as yellow, and collagen as red. To quantify the extent of fibrosis, Imaged software (National Institute of Health, Bethesda, MD, USA) was used. Briefly, a macro previously designed to calculate fibrosis was modified for use with our software, and was expanded for utilization in adipose tissue.

[0144] Mitochondrial bioenergetics

[0145] High-resolution respirometry: Mitochondrial respiration was analyzed in RG, WG, LV, liver, brain, eWAT and iWAT using high-resolution respirometry (Oroboros Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria). Each chamber was set to 37°C, with the exception of LV which was set to 25°C, maintained constant stirring at 750rpm, and contained 2 mL mitochondrial respiration buffer (MiRO5 - 0.5 mM EGTA, 3 mM MgCI2 H2O, 60 mM potassium lactobionate, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 20 mM taurine, and 1 g L— 1 fatty acid-free BSA; pH 7.1 ).

[0146] Measurements of mitochondrial respiration were performed using saponin-permeabilized red gastrocnemius and white gastrocnemius (PmFB) in the presence of 5 pM blebbistatin, left ventricle PmFBs in the absence of 5 pM blebbistatin, in liver and cortex strips, and in finely minced eWAT and iWAT in the presence of 50 pM saponin. In all tissues, mitochondrial respiration following the addition of 5 mM pyruvate + 2 mM malate, 5 mM ADP (titrated from 0.25 pM to 10 mM in PmFB), 10 mM glutamate, 10 mM succinate and 10 pM cytochrome C. Respiratory control ratios (RCR), which reflect mitochondrial coupling state, were determined for all experiments. Mitochondrial respiration was normalized to tissue dry weight within RG, WG and LV, was normalized to tissue wet weight in liver and brain, and was normalized to 1 ) tissue wet weight and 2) total cell number in eWAT and iWAT. Total cell number was calculated using an estimate of cell volume derived from two-dimensional histology images in combination with the relative density of fat (1 g / 1.0869 cm3). These calculations were carried out under the assumption of adipocyte spherical structure. The addition of cytochrome c did not increase respiration >10 % in any experiment.

[0147] Mitochondrial reactive oxygen species emission: Mitochondrial H2O2 emission was determined fluorometrically (Lumina, Thermo Fisher Scientific, Waltham, MA, USA) within RG, WG, LV and eWAT. Briefly, experiments were conducted at 37°C in RG, WG and eWAT samples, and at 25°C in LV samples. Tissues were placed in a constantly stirring cuvette containing Buffer Z (105 mM K-MES, 30 mM KCI, 1 mM EGTA, 10 mM KH2PO4, 5 mM MgCI2, 5 pM glutamate, 5 pM malate, and 0.5% FFA- free BSA, pH 7.1 ). In eWAT tissue, experiments were conducted in the presence of 10 pg / mL digitonin, 1 U / mL horseradish peroxidase, 40 U / mL superoxide dismutase (SOD), and 10 pM Amplex Red (Invitrogen, Carlsbad, CA). Mitochondrial H2O2 emission was determined in the presence of 20 mM succinate. In PmFB, experiments were conducted in the presence of 1 U / mL horseradish peroxidase, 40 U / mL superoxide dismutase (SOD), and 10 pM Amplex Red (Invitrogen, Carlsbad, CA). Mitochondrial H2O2 emission was determined in the presence of 20 mM succinate, and 100 pM ADP. Prior to the experiments, raw fluorescence was calibrated to a standard curve using known concentrations of H2O2. Mitochondrial H2O2 emission data was normalized to fibre dry weight in PmFB and to wet weight in eWAT.

[0148] Western blotting

[0149] Standard Western blotting procedures using whole muscle crude homogenates from RG samples were conducted. Briefly, samples were diluted to 1 pg / pL, and loaded as per manufacturer’s instructions for separation with standard SDS-PAGE gels, transferred to polyvinyl difluoride membranes, blocked, and incubated with corresponding primary and secondary antibodies. The following commercially available antibodies were used: Akt (1 :2000, Cell Signalling cs4691 L), p-AktS473 (1 : 1000, Cell Signalling cs9271 s), AMPK (1 : 1000, Cel Signalling 2532), p-AMPKThr172 (1 :1000, Cell Signalling 2535), ERK1 / 2pp44 / 42 (1 :1000, Cell Signalling cs4695s), p-ERK1 / 2pp44 / 42Thr202 / Tyr204 (1 : 1000, Cell Signalling, cs9101 s) , eEF2 (1 : 1000, Cell Signalling cs2332), p-eEF2Thr56 (1 : 1000, Cell Signalling cs2331 ), mTOR (1 :1000, Cell Signalling cs2972s), p-mTOR (1 :1000, Cell Signalling cs2971 s), P70S6 (1 : 1000, Cell Signalling 9202) and p-P70S6Thr389 (1 : 1000, Cell Signalling 9234). Each membrane was visualized using enhanced chemiluminescence (PerkinElmer, Voston, MA, USA), and quantified by densitometry (FluorChem HD2 Alpha Innotech imager; Fisher Scientific, Waltham, MA, USA). Ponceau staining was used as a loading control.

[0150] Tissue protein synthesis

[0151] Labelled water provision

[0152] A subset of mice consuming each diet received one intraperitoneal injection of 99% deuterium oxide at 0.015 mL / g body mass, and were provided ad libitum access to water enriched with 4% deuterium for the duration of the 7 day assessment period. Following this period, tissues were excised, as

[0153] Serum free [2H]-alanine enrichment

[0154] Serum [2H]-alanine enrichments were measured using GC-MS (Agilent 5973N MSD and 6890N GC). Serum samples were deproteinized, purified, and derivatized, before analysis by GC-MS as previously described (Holwerda et al., 2018; Petrick et al., 2023). Serum free alanine mass isotopomers (M and M + 1 ) were determined using selective ion monitoring at m / z 232 and 233. Standard regression curves were used from a series of known standard enrichment values against the measured values to assess the linearity of the mass spectrometer and to account for any isotope fractionation.

[0155] Protein-bound [2H]-alanine organ tissue enrichment

[0156] For measurement of protein-bound [2H]-alanine enrichments, tissues (heart, white gastrocnemius, red gastrocnemius, liver, diaphragm, kidney, lung, pancreas, intestines, brain) were freeze-dried and weighed . Skin and bone were weighed and crushed . Subsequently, ice-cold 2% perchloric acid was added at a ratio of 35 times dry weight (for freeze-dried tissues) or 7 times wet weight (for skin and bone). All samples were sonicated using ultrasonic disintegration (Soniprep; MSE) for 4 times of 15 sec each. Samples were then centrifuged at 3500 rpm for 15 min at 4°C. The protein pellet was washed with three additional 1 mL washes of 2% perchloric acid and the supernatant discarded. The pellet was then hydrolyzed overnight (16 h) in 6 M HCI at 120°C. The hydrolyzed protein fraction was dried under a nitrogen stream while being heated at 120°C for 2-3 h. After being dissolved in acetic acid solution, samples were purified, derivatized , and measured by a gas chromatography mass spectrometer (GC-MS; Agilent 5973N MSD and 6890N GC, Little Falls, NY, USA) (Petrick et al., 2023). Protein-bound alanine mass isotopomers (M and M + 1 ) were determined using selective ion monitoring at m / z 232 and 233. A series of known standards were applied to assess linearity of the mass spectrometer and to control for the loss of tracer.

[0157] Calculations

[0158] Tissue FSR was determined by the incorporation of [2H]-alanine into organ proteins, using free [2H]- alanine enrichment in serum as the precursor. FSR was calculated using first-order kinetics equation, as follows : where f is the cumulative fractional synthesis, which was determined by dividing the protein-bound [2H]-alanine enrichment in the organ samples by the free [2H]-alanine enrichment in the serum samples, and t represents the time from D2O ingestion until tissue collection. One littermate mouse housed in identical conditions consuming the standard diet (7% EAA + 11 % NEAA) was used to provide unlabelled control samples for background correction.

[0159] Transcriptomics

[0160] RNA samples were sent to the McMaster Genomics Facility where an RNA quality check, poly A enrichment, library prep and quality check were performed as previously described (50). The samples were then run in an Illumina Nextseq P2, 2x50bp sequencing run. FastQC and MultiQC were used for quality control of raw data. DESeq2 was applied for detection of differentially expressed genes (DEG) , and genes with counts lower than 10 were filtered out in any sample. Principal component analysis (PCA) was performed by using variance stabilizing transformation (VST) data through DESeq2. We used Enricher to find GO enrichment terms (https: / / maayanlab.doud / Enrichr / ) and searched for genes to create heatmaps and KEGG library (https: / / www.genome.jp / kegg / kegg1 b.html). The results were illustrated in a gene-concept network diagram using the cnetplot package (https: / / bioconductor.org). Transcriptomic analyses were performed using the Linux system, R and RStudio software version 2024.04.2+764.

[0161] Serum metabolite profile

[0162] Serum FFA, TAG, GDF15 and FGF21 were determined as previously reported using commercially available kits (Petrick et al., 2023; Wang et al., 2023). A basic global metabolomics analysis was conducted at The Metabolomics Innovation Center (University of Alberta) using a combination of untargeted and targeted high-performance chemical isotope labeling LC-MS metabolomics as previously reported (Zhao et al., 2019).

[0163] Proteomics

[0164] Liquid chromatography-mass spectrometry (LC-MS) was used quantify protein abundance (proteomic analysis) in whole muscle homogenates using data dependent analysis as previously reported (Frangos et al., 2025). Briefly, the Vanquish Neo UHPLC system was combined with an Orbitrap Exploris 240 mass spectrometer using the Easy-Spray source for nanoLC-MS-based protein identification. Protein identification was established using the false discovery rate (FDR), while label-free quantification (LFQ) was used to determine the relative abundance of proteins across samples.

[0165] Statistics

[0166] Statistical analyses were completed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). One-way ANOVA was used to compare differences between groups, and when significance was obtained, Tukey’s honest significant difference (LSD) post hoc test was used. In experiments involving comparison between lean or obese groups of Con vs 18% EAA animals, an unpaired students t-test was used. For mitochondrial bioenergetics data, the apparent Km was estimated with Michaelis-Menten Kinetics, and maximal respiration (Vmax) was specified as the highest respiration value directly determined. RNAseq data was analyzed using R (https: / / www.r-project.org / ), where principal component analysis was performed using vegan package, and heatmaps were generated using the pheatmap package (https: / / github.com). R scripts may be provided upon request. Data are expressed as scatter plots of individual values, with mean ± SD superimposed. Significance was set at p < 0.05 for all data with the exception of RNAseq, where significance was set at p < 0.1.

[0167] EXPERIMENT 1

[0168] Impact of dietary changes in non-EAA and EAA abundance on body morphology.

[0169] The effect of consuming a diet devoid of non-EAA was tested, but matched to a control diet for either EAA content (7% EAA) or total protein (18% EAA) on body weight and key metabolic tissue mass / morphology. While both non-EAA depleted diets caused weight loss, the higher abundance of EAA within the 18% diet caused an even more marked, and rapid, weight loss (Fig. 1A, B), resulting in a surprising -25% reduction in weight loss over the 14-day intervention (Fig. 1 B). While decreased caloric intake during the first week likely contributed to weight loss with non-EAA depletion (Fig. 1 C, E), caloric intake was not different over the second week of the intervention despite continued weight loss (Fig. 1 D, F; Fig. 8A, B), and occurred in the absence of changes in fecal energy content (Fig. 8C, D). Combined, these data show the pronounced weight loss with these NEAA depleted diets cannot be explained by decreased food intake or compromised intestinal absorption.

[0170] An increased energy expenditure and fat oxidation could contribute to the rapid weight loss and was examined. During the first week, while indexes of energy expenditure (Vo2 and heat production) were not altered (Fig. 1 G, I, K), there was a shift observed towards greater fat oxidation (i.e. decreased RER) following the 18% EAA diet (Fig. 1 M). During the second week, while indexes of absolute energy expenditure (including Vo2 and heat production) remained constant (Fig. 8H-J), given the marked reduction in weight, relative Vo2 and heat production were increased in mice consuming non-EAA depleted diets (Fig. 1 H, J, L, N).

[0171] Given the rapid weight loss and apparent increase in whole-body fat oxidation (lower RER 1 st week, higher Vo2 and heat 2nd week) , the body composition during the dietary intervention was examined. While there were no observable changes in body composition during week 1 (Fig. 9A-E), after 2 weeks whole body fat (-70%) and lean (-10%) abundance were decreased, particularly in mice consuming the 18% EAA (Fig. 1 O-S). In further support, eWAT tissue weight (Fig. 1T) and CSA (Fig. 1 U) were greatly reduced in both experimental groups, particularly in the 18% EAA group (-50-75% lower). While prominent within fat tissue, these changes were not specific to adipose, as similar responses were observed in liver tissue weight (Fig. 1 V) and CSA (Fig. 1W).

[0172] Concluding, while depletion of non-EAA in the diet decreased adiposity, increasing EAA content of the diet dramatically decreased the abundance of whole body and visceral fat. These findings occurred with slight reductions in lean mass, indicating the impact of the dietary intervention was primarily on fat mass.

[0173] EXPERIMENT 2

[0174] Impact on cardiometabolic health

[0175] Fat loss is known that it may improve glycaemic control, there is also a concern that rapid liberation of non-esterified fatty acids (NEFA) could manifest in lipotoxicity and negative health outcomes similar to the existing models of lipodystrophy. Therefore, various indexes of cardiometabolic health were examined. While serum NEFA was decreased following the consumption of both non-EAA depleted diets, serum TAG was reduced following consumption of the 18% diet (Fig. 2A, B), suggesting the absence of lipotoxicity. Additionally, while serum GDF15 was not altered in any group, circulating FGF21 was increased in both intervention diets (Fig. 2C, D), a response previously associated with improved cardiometabolic health. In support of this, while fasting blood glucose and indices of insulin sensitivity were not significantly affected by weight loss, whole body glucose tolerance was improved in the 18% EAA group (Fig. 2E-H) despite reductions in lean mass (Fig 1 R), skeletal muscle mass (Fig. 2I) and rates of protein synthesis (Fig. 2J). Notably, both 7% and 18% diets significantly reduced all normalized tissue weights (Fig. 12A-E). In contrast, the 7% EAA diet reduced rates of muscle protein synthesis in the kidney, lung and skin, while the 18% EAA diet significantly reduced rates of muscle protein synthesis in all tissues compared to control animals, and in many tissues compared to the 7% EAA group (Fig. 13A-K). While muscle mass and insulin signaling were linked with improved glucose homeostasis and improved glucose tolerance coincided with marked reductions in fat mass (Fig. 1 O, Q, T) and liver adiposity (Fig. 2K). While eWAT fibrosis was observed to be increased in 18% EAA mice (Fig. 2L), without being bound by any particular theory, it was believed that this may represent an artifact of rapidly shrinking adipocyte size, as demonstrated by the increasing linear regression slope when comparing cells per field of view versus percent fibrosis (Fig. 11 C, D). This trend was also seen in iWAT tissue (Fig. 11 E, F).

[0176] Given the relationship between body fat loss and improvements in cardiovascular risk factors, cardiac morphology was examined. Similar to skeletal muscle, total mass, rates of protein synthesis and CSA were all reduced in the heart in animals consuming both diets, however the rate of protein synthesis was reduced in the 18% EAA group (Fig. 2M-P). While the reduction in LV CSA likely may have contributed to reduced posterior wall thickness, indices of cardiac function were not altered (Fig. 2Q-U, Table 3).

[0177] Table 3: Additional echocardiography data: Values represent the mean ± SD (P < 0.05); n = 14-15 animals / group. Statistical analysis: one way ANOVA with Tukey HSD post hoc test for multiple comparisons. Combined, the data shows the diet-induced weight loss is associated with positive cardiometabolic health outcomes, including improved glucose tolerance and reduced hepatic lipid accumulation.

[0178] Given the relationship between oxidative phosphorylation and redox stress on cellular function in diverse tissues mitochondrial bioenergetics, including submaximal ADP supported respiration and H2O2 emission were examined (Fig. 3A,B depict representative traces). Consumption of 18% EAA died did not alter mitochondrial respiration or H2O2 emission in permeabilized muscle fibres from either skeletal muscle (Fig. 3C-G, Fig. 14A-E) or LV (Fig. 3H-L), or respiration in permeabilized liver or brain tissue (Fig. 3M,N). While respiration per milligram adipose tissue weight was higher following 18% EAA (Fig. 30, Fig. 14F), this was caused by smaller adipocyte size concentrating mitochondrial content, as respiration was lower when normalized to cell number (Fig. 3P, Fig. 14G).

[0179] Combined, the data shows that consuming a diet high in EAA and devoid of non-EAA does not alter mitochondrial bioenergetics in diverse tissues.

[0180] EXPERIMENT 3

[0181] Interrogating tissue-specific changes

[0182] Given the marked reduction in body weight, adiposity, hepatic lipid accumulation and improved glucose tolerance in mice consuming a diet high in EAA and devoid of non-EAA without alterations in mitochondrial bioenergetics, alternative mechanisms-of-action were investiged. The transcriptome responses was analyzed following 4 days of 18% EAA consumption in a visceral adipose depot (eWAT), the liver and an oxidative skeletal muscle (red gastrocnemius muscle), as this time point represents an early response before overt weight loss could confound interpretations. While the 18% EAA diet did not alter gross expression patterns within eWAT (Fig. 4A), there were 1646 differentially expressed genes (DEG; Fig. 4B), including 854 down-regulated genes. While all DEF genes highlighted broad pathways pertaining to metabolism (Figure 15), when only considering down- regulated DEF, CNET analysis highlighted 8 genes involved in proteostasis (Fig. 4C) and several genes involved in amino acid metabolism. Given the short duration of the intervention, filtering DEF genes to include only those reduced >1 fold to be more conservative, KEGG enrichment and Gene Ontology (GO) analysis highlighted broad metabolism, particularly fatty acid metabolism (Fig. 4C, D), an interpretation supported through the identification of genes affected by the top 10 GO enrichment processes, which included decreased expression of genes involved in lipid storage / synthesis (Fig. 4E; Fig. 15A). Surprisingly, a similar pattern was observed in the liver, as despite the modest changes in genes (Fig. 5A, B), the most differentially expressed down-regulated genes were associated with fatty acid metabolism pathways and genes (Fig. 5C-F; Supplemental Fig. 8B).

[0183] Given the consistent changes in metabolic processes, particularly fat metabolism, within eWAT and liver, these tissues were further analyzed for conserved gene expression patterns. Specifically, 102 similarly down-regulated genes were identified (Fig. 6A) which clustered within several metabolic processes, including carbon, amino and fatty acid metabolism (Fig. 6B). Additionally, the top 10 biological pathways identified in GO analysis clustered around fatty acid metabolism (Fig. 6C). This included Acly, Fasn, Acaca and Elovl6 which displayed marked reductions in both adipose and liver (Fig. 6 D, E) and are all involved in fatty acid synthesis (Fig. 6F). In contrast to the consistent effects of 18% EAA consumption on genes / pathways within eWAT and liver, skeletal muscle was largely unaffected (Fig. 15C,D).

[0184] Combined, these transcriptional data suggest consumption of the 18% EAA diet decreased genes involved in lipid storage within eWAT and liver, supporting the phenotype of fat loss.

[0185] EXPERIMENT 4

[0186] Clinical translation

[0187] Since obesity is a significant risk factor associated with the development of cardiovascular disease it is becoming increasingly important to develop effective treatment paradigms that can assist in reducing adiposity. Treatments contemplated in accordance with the invention on lean animals with diets devoid of dietary NEAAs, with or without a compensatory increase in EAA intake, were shown to significantly reduce whole body adiposity and improved glucose homeostasis without negative off- target effects on indices of cardiac function. This indicates treatments in accordance with the invention may benefit subjects not being overweight or obese, but the surprising advantageous effects prompted to study the effect in an obese model. Hence, the therapeutic potential in a model of hyperphagia- induced obesity (Fig. 7A) was assessed. To more accurately assess the functional relevance of this diet on clinical populations, both male and female mice across a diverse age range (4-12 months) were included and block randomized mice to consume either a control or 18% EAA diet for 2 weeks. Similar to lean animals, obese animals consuming the 18% EAA diet lost a significant amount of body weight by day 2 (Fig. 7B, C), and while caloric intake (Fig. 7D) and body weight (Fig. 7E) were significantly lower following 1 week, weight loss continued over the second week despite unaltered caloric intake (Fig. 7F-G; Fig. 16A-C), showing that overall changes in food intake were not solely responsible for the observed weight loss.

[0188] To gauge where the weight loss was occurring, measurements of diverse tissue weights were obtained. While iWAT (Fig. 7H) and liver (Fig. 7J) were significantly lower, heart weight was unchanged (Fig. 7I; additional tissues weights in Fig. 16G-J), once again indicating adipose tissue and liver as key tissues affected by the dietary-intervention.

[0189] Given the relationship between hyperlipidemia and reduced glucose handling, fasting glucose and performed measurements of fuel utilization was further analyzed. Fasting blood glucose, Vo2, RER, and heat production when expressed per unit body weight were all significantly reduced in 18% EAA mice (Fig. 6K-O, Fig. 16 D-F). While mitochondrial respiration was unaltered in diverse tissues (Fig. 6P-R, Fig. 17A-C,F,H), maximal mitochondrial H2O2 emission was reduced following 18% EAA dietary consumption within both the heart (Fig. 7S, T) and eWAT (Fig. 17G). Given the reduction in mitochondrial H2O2within the heart, indices of LV cardiac function were measured. Consumption of 18% EAA decreased PWT (Fig. 7V), and increased EDV (Fig. 7W), indicative of pathological hypertrophy reversal. These changes (larger EDV with no change in ESV) manifested in an increase in SV (Fig. 7X) and CO (Fig. 7Y) without any changes in other indices of cardiac function (Fig. 19A-E). Surprisingly, while HR correlated with SV in all mice (Fig. 7Z), the slope of the regression line was lower in the 18% EAA group, indicating a greater SV for these animals at a higher HR, further suggesting improvements in cardiac “relaxation”. In a separate analysis, LV echocardiogram data were compared before and after the 2-week dietary intervention, and the significant findings were preserved (Fig. 18A-H). These changes in indices of cardiac function did not manifest in any significant improvements in LVP max (Fig . 7Zi), LVP min (Fig. 7Zii) or MAP (Fig. 7Ziii) (see additional measurements in Fig. 19F-O).

[0190] Concluding, the data shows that a diet devoid of NEAA and providing 18% EAA results in significant fat loss in obese mice and improved cardiometabolic health, including reduced fasting blood glucose levels and indices of improved cardiac function.

[0191] DISCUSSION OF EXPERIMENTS 1-4

[0192] The observations of experiments 1 -4 demonstrate that depletion of NEAA from the diet with or without a compensatory increase in EAA induces i) a staggering -25% reduction in body weight, primarily attributed to ii) a loss in visceral adiposity and Hi) increased whole body fat oxidation, which manifested in improved cardiometabolic risk profiles, including iv) reduced serum lipid profiles, v) improved glucose tolerance, vi) and reduced hepatic lipid abundance without any detectable negative or off-target effects. While changes in mitochondrial bioenergetics could not explain the changes in lipid metabolism, transcriptomics suggested reductions in lipid synthesis within WAT and liver could contribute to the observed phenotype of these mice. Highly advantageously, a diet devoid of NEAA and fortified with EAA displayed similar health benefits in a model of hyperphagia-induced obesity, including dramatic weight loss, improved glucose homeostasis, and increased cardiac output, validating the translational utility of this diet. Together, the results from experiments 1-4 show substantial cardiometabolic benefits of diets devoid of NEAA and fortified with EAA, a dietary intervention that could have a remarkable potential i.a. to reverse the growing obesity epidemic.

[0193] Mechanistically, the surprising -25% reduction in adiposity is likely mediated by changes in energy metabolism, as the acute decrease in food intake only lasted a few days, whereas weight loss persisted over the second week while food intake was normalized. The transient reduction in caloric intake could only account for minimal amount of fat loss, and given the observed significant reduction in body weight in both groups, it is clear both ablation of NEAAs and increased EAA intake are contributing factors. Previous work has implicated FGF21 as a possible contributing factor for protein deficient diet-mediated weight loss given the marked rise in serum FGF21 during weight loss, the inverse relationship between FGF21 and adiposity, and ablation of FGF21 preventing low protein diets from altering basal energy expenditure and body weight. While in the present experiments 1 -4 a protein deficient diet (7%) caused weight loss in association with an increase in circulating FGF21 , fortification of the diet with EAA dramatically decreased weight and fat mass without further increasing FGF21. Of note, animals consuming the 18% EAA diet were observed to display a conserved downregulation of many genes within the liver and adipose tissue, including fasn, acly and elovl6, indicating a separate mechanism of action through which fatty acid synthesis is inhibited. Of these genes, expression of fasn and acly is inhibited by the presence of FGF21 , as FGF21 KO mice exhibited significantly increased fasn expression following administration of a diet void of leucine compared to control mice. While data show a key role for FGF21 in mediating the health benefits of protein restricted diets, in the present experiments 1 -4 FGF21 was observed to be similar between the 7% and 18% groups despite the greater health benefits while consuming the 18% EAA diet. Without being bound by any particular theory, the authors believe that a higher abundance of EAA may affect cellular mechanisms as leucine enrichment has previously been shown to increase mTOR activation, a known regulator of SREBP-1 which increases expression of elovl6. Additionally, unregulated mTOR stimulation in the absence of NEAA availability may initiate futile amino acid recycling as an energetically demanding process, as mTOR signaling simultaneously activates protein synthesis and concomitantly protein degradation by the ubiquitin proteasome system. While transcriptional networks show proteostasis, amino acid metabolism and ER-stress following the 18% diet, rates of protein synthesis and skeletal muscle mTOR signaling were not altered indicating the necessity of NEAA to optimize protein synthesis rates in diverse tissues.

[0194] Models of lipodystrophy are thought to manifest in negative health outcomes as a result of the excessive delivery of fatty acids to peripheral tissues. However, in the present experiments 1 -4 the almost complete ablation of adiposity corresponded to reductions in serum and hepatic lipid accumulation and improvements in cardiometabolic health suggesting an increase in fat oxidation occurred. There were increases in VO2 and heat production over the second week of the intervention when normalizing to body weight, and these observations were not different between the two intervention diets. Surprisingly, these indices of energy expenditure were not altered in the first week despite weight loss, suggesting the absence of dramatic changes in whole-body energy expenditure. However, it has been estimated that adipose tissue only contributes ~4% to basal energy expenditure in humans, and therefore a consistent increase in energy expenditure specifically within this tissue may be difficult to measure at the whole-body level. As a result, without being bound by any particular theory, it is likely the EAA enrichment-mediated changes in adiposity are due to an increased energetic stress within adipose and liver tissues. Particularly, a futile cycle where lipids are broken down and partially re-esterified leading to increased energetic stress within these tissues may offer insight into the mechanism at play. For instance, in adipose tissue up to 85% of fatty acids broken down from TAG are at least partially re-esterified in an energetically costly process that requires 4 moles of ATP for every mole of re-esterified TAG. Notably, it has been observed that enrichment with ketogenic EAAs may facilitate a rapid increase in lipolysis independent of changes in UCP1 -dependent thermogenesis. Furthermore, enrichment with ketogenic EAAs repressed liver expression of fasn and scd1 in obese mice in a process likely mediated by suppression of SREBP-1 c, which combined with the present transcriptomic data suggests a mechanistic link between EAA enrichment and futile lipid cycling in liver and adipose tissues. While the health benefits to reduced adiposity have a well-established role in cardiometabolic risk reduction, in the present experiments obese animals fed an 18% diet also displayed an increase in cardiac output. While the mechanisms remain to be investigated, given the increase in EDV and reduction in end-systolic posterior-wall thickness without changing blood pressure, this diet manifests in a phenotype indicative of a reversal of diabetic cardiomyopathy, a condition associated with pathological remodeling in the absence of changes in blood pressure.

[0195] Concluding, the present invention shows that a NEAA depleted diet, with or without additional EAA intake, strongly decreases adiposity with concomitant cardiometabolic health benefits, including improved glucose homeostasis and reduced serum and hepatic lipid accumulation. These changes in body composition and cardiometabolic health benefits translated to a model of obesity and were further accompanied by improvements in cardiac morphology / function. A diet devoid of NEAA with additional EAA intake may be applied as a practical nutritional strategy to effectively improve body composition and prevent or treat cardiovascular derangements, such as associated with obesity and being overweight.

[0196] EXPERIMENT 5

[0197] In this human clinical nutrition experiment the impact of ingesting a (very) low protein intake diet supplemented with various amounts of EAA and NEAA, in accordance with the invention, is assessed. Compositions used are analogous to the compositions used in Experiments 1 -4, adapted to the practical possibilities when designing a human trial. This experiment is conducted to assess tolerability of these diets with differing EAA / NEAA ratios and to assess the impact on body weight, body composition, and risk factors for chronic metabolic disease. As a first human clinical trial this is performed over a relative short time frame (10 days) studying the health benefits of lowering NEAA and / or increasing EAA content of the diet.

[0198] The primary objective of the study is to determine the influence of a diet lacking NEAA, with or without being replaced by additional EAA, on skeletal muscle protein synthesis rates in humans. The secondary objective is to assess the impact of diets lacking NEAA (with or without being replaced by additional EAA) on skin protein synthesis rates, muscle and organ mass, resting energy expenditure, blood pressure, heart rate, skeletal muscle mitochondrial respiration, plasma and urine profiles, and skeletal muscle signaling pathways.

[0199] MATERIALS AND METHODS FOR EXPERIMENTS 5

[0200] A parallel design in healthy young male participants is used. The total duration of the study for each subject is ~14 days. In total, 45 healthy young men will complete the study. All subjects consume a standardized low protein diet (<2% energy as protein) supplemented with various amounts of EAA and NEAA in the form of free amino acid drinks. Subjects are randomly assigned to one of three groups: a conventional diet group (standardized diet supplemented with 7% energy as EAA + 10% as NEAA), a diet lacking NEAA (supplemented with 7% EAA + 0% NEAA), or a diet lacking NEAA and replaced by additional EAA (supplemented with 17% EAA + 0% NEAA, isonitrogenous with conventional diet) . The dietary intervention will last 10 days (Figure 20). Each participant participates in a screening session (~ 1 h), a D2O dosing day (one day prior to beginning the dietary intervention, ~ 4h), an experimental test day at beginning the dietary intervention (~ 2h, Test Day 1 , Day 1 ), an experimental test day halfway through the dietary intervention (~ 0.5h, Test Day 2, Day 6), and an experimental test day after completing the intervention (~ 2.5h , Test Day 3, Day 11 ). Throughout the dietary intervention period, participants consume one 20 mL dose of 70% deuterium oxide daily. Participants will collect a saliva sample daily. Physical activity will be tracked with accelerometers prior to and during the dietary intervention, and habitual physical activity will be recorded. The study design is schematically outlined in Figure 20.

[0201] Population (base)

[0202] In total, forty-five healthy (BMI 22 - 30 kg / m2) young (age: 18 - 35 y) males participate in the present study. The nature and the risks of the experimental procedures are explained to all subjects before their informed consent will be obtained..

[0203] Inclusion criteria

[0204] In order to be eligible to participate in this study, a subject must meet all of the following criteria:

[0205] • Male

[0206] • Age between 18 and 35 y inclusive

[0207] • BMI between 22 and 30 kg / m2

[0208] • Non-smoker

[0209] • Having given informed consent

[0210] Exclusion criteria

[0211] A potential subject who meets any of the following criteria are excluded from participation in this study:

[0212] Participating in a structured (progressive) exercise program Smoker

[0213] Diagnosed Gl tract disorders or diseases

[0214] Diagnosed musculoskeletal disorders

[0215] Diagnosed metabolic disorders (e.g. diabetes)

[0216] Cardiovascular disease

[0217] Hypertension (blood pressure above 140 / 90 mmHg) Donated blood 3 months prior to test day

[0218] Use of any medications known to affect protein metabolism (i.e. corticosteroids, nonsteroidal anti- nflammatories).

[0219] Chronic use of gastric acid suppressing medication Chronic use of anti-coagulants

[0220] Any intolerance to foods included in the standardized diet intervention Any implants that would be a contra-indication for performing an MRI scan

[0221] Sample size calculation

[0222] The sample size calculation was based on the primary outcome of differences in fractional synthetic rates (FSR) between the different interventions. The calculation was performed in GPower version 3. 1.9.6 (Dusseldorf, Germany). Based on previous published data we expect the standard deviation for daily muscle protein FSR to be approximately 0.30% d-1 in all groups. Based on a previous study examining the effect of additional leucine supplementation on top of normal protein intake, the difference in muscle protein FSR between interventions was 0.36% d-1. Since this previous study was an acute intervention in a laboratory setting, a smaller change was expected between groups in a chronic intervention due to lifestyle factors such as habitual physical activity. Therefore, we expect a change of 0.30% d-1 between dietary interventions in a chronic 10-day period. This is a -20% difference when expressed as relative difference from the control group, which is considered a clinically relevant difference between interventions.

[0223] Using these values, a one-way ANOVA a-level of 0.05 and 80% power, the calculations show that 15 participants per group would be adequate to observe this difference in daily FSR between diets. Since the study will include 3 groups, the total amount of subjects that will need to complete the study will be 45.

[0224] In case a participant drops out during the interventional trials, the participant will be replaced with a newly randomized participant. The potential drop-out rate is expected to be the same in all three groups and would maximally amount 10% per group. Therefore, a maximum of 51 participants are expected to be randomized (15 + 10% = 17 x 3 groups = 51 ). Taking into account an expected screening exclusion rate of maximally 20% , a total of 64 participants are expected to be screened. The study is completed when 45 participants have successfully completed the study.

[0225] TREATMENT OF SUBJECTS

[0226] Investigational treatments

[0227] Experimental diet

[0228] All subjects will consume a standardized low-protein diet (<2% energy as protein, 70% energy as carbohydrates, 28% energy as fat) supplemented with various amounts of EAA and NEAA in the form of free amino acid drinks. Participants will be randomly assigned to one of three intervention groups: 1 ) a conventional diet (standardized diet supplemented with 7% energy as EAA + 10% as NEAA), 2) a diet lacking NEAA (supplemented with 7% EAA + 0% NEAA), or 3) a diet lacking NEAA and replaced by additional EAA (supplemented with 17% EAA + 0% NEAA, isonitrogenous with conventional diet). The diet will be consumed for 10 days. A prolonged diet very low in protein does not lower fat-free mass (diet composed of 5% energy from protein) or lower muscle protein synthesis rates (diet composed of 9% energy from protein), suggesting that a very low protein intake does not compromise muscle mass maintenance. However, it is anticipated that dietary changes in amino acid intake (the interventional drinks provided) may still be capable of having an effect, e.g. influencing muscle protein synthesis over 10 days. Changes in muscle protein synthesis and / or net protein deposition have been identified following 6 days of whey protein supplementation, 14 days of leucine supplementation, and 14 days of consumption of animal vs. plant-based proteins.

[0229] Table 4 - AA composition for EAA powder and NEAA powder (per 100g powder) All standardized diets will be proportioned to meet the subjects’ energy requirements which will be calculated with the revised Harris and Benedict equation for young individuals, with a physical activity index of 1.5 - 1.7, depending on physical activity levels. Physical activity levels will be assessed on the screening visit using the International Physical Activity Questionnaire (IPAQ). Based on the information on the questionnaire, physical activity levels will be classified as low, moderate, or high. This will correspond to using a physical activity index of 1.5 (low physical activity), 1.6 (moderate physical activity), or 1.7 (high physical activity). In the conventional diet group, 17% energy as protein equates to -1.6-1.8 g / kg body mass I day of protein intake (plus the <2% energy as protein in the standardized low-protein diet), which is representative of young healthy Dutch males. The standardized low protein diet will be created using commercially available foods and provided by the university personnel. The diet has been composed in consultation with a dietician to meet the requirements for a balanced intake of macronutrient foods, and has previously been used in a research study. All products required for the standardized diet will be bought at regular supermarkets, with the exception of the specialized products which are low in dietary protein (e.g. low protein pasta, bread, candy bar). The product low in protein will be order at a specialized supplier of low-protein productions (e.g. Sorgenta / Tefa, the Netherlands). All food products will be stored in an appropriate closet or fridge in the “dietary kitchen” at the Department of Human Biology. Small variations in the standardized diet are possible (for example, extra protein-free candy bars as snacks), however, the main meals will be kept the same throughout the test period.

[0230] Foods will be weighed for each participant individually. On Test Day 1 , participants will be provided with the first 5 days of meals (breakfast, lunch, dinner, beverages, snacks) in take-home packages with meal planning and instructions of how to prepare the meals , and labelled appropriately with the storage conditions of the foods. The free amino acid drinks will be provided to consume 4 times daily - at breakfast, at lunch, at dinner, and before sleep. The free amino acid mixtures will be manufactured by a commercial nutritional company (PYC Laboratoire, France) with AA composition identical to that of milk protein (20). AA compositions are displayed in the table below. Two mixtures will be manufactured - one of EAA and one of NEAA. The amount of each powder provided to participants will be adjusted based on which group they are randomized to and their individual dietary caloric requirements. In the group with NEAA removed and not replaced by additional EAA (i.e. consuming 7% EAA and 0% NEAA), the calories / macronutrients will be replaced by additional carbohydrates (commercially available maltodextrin administered in powder form, (AMACX, similar to the EAA and NEAA drink in power form). All drinks will have 1 g of Stevia (Canderel, Sligro) added. All powder mixes will be provided to participants in sterile 75 mL containers as powders, with instructions on how to pour and mix in an electric shaker bottle provided (XXL Nutrition, the Netherlands), how much water to add, and how to prepare the drinks prior to the appropriate time of ingestion.

[0231] Dietary compliance will be assessed by regular contact with the investigators and instructing the participants to return any un-consumed foods (on Test Day 2, Day 6 of the intervention; and on Test Day 3, Day 11 of the intervention). Participants will be provided with the remaining 5-days of meals on Test Day 2.

[0232] Diet and physical activity

[0233] All subjects will be asked to avoid caffeine and alcohol before each of the Test Days, and to consume a standardized dinner meal the day before Test Day 1. This standardized meal is an ‘Aviko maaltijdpannetje’ a ‘Mondelice vanille dessert’ and a ‘Liga Evergreen’ biscuit as evening snack, and will be provided to the volunteers in a thermal bag at the end of the preceding session to take home in preparation for the Test Day (Thus at the end of the D2O Dosing Day). The standardized meal will not be provided before the other two test days, because participants will be consuming the interventional diet. Furthermore, volunteers will be instructed to refrain from any sort of heavy physical exercise for the 3 days before each Test Day. Additionally, subjects will be asked to record their food intake for the 3 days before Test Day 1 and Test Day 3 in a food diary that will be provided during the screening day (for Test Day 1 ) or on Test Day 2 (for Test Day 3). Participants will also be asked to record their physical activity performed the 3 days prior to Test Day 1 and Test Day 3 in an activity log that will be provided on the screening day (for Test Day 1 ) or on Test Day 2 (for Test Day 3).

[0234] Oral deuterium oxide (tracer) ingestion

[0235] The on-going and continuous processes of tissue protein synthesis and breakdown ultimately dictate tissue adaptations, including changes in muscle mass, and is influence by dietary intake. Data on human skeletal muscle protein synthesis has commonly been obtained with the use of stable isotope amino acid tracer techniques. Deuterated water (2H2O) ingestion is a stable isotope method that is commonly applied to measure tissue protein synthesis rates over days to weeks in human study participants. The approach for this study requires that participants ingest deuterated water on the dosing day for the duration of the experimental period.

[0236] Assessments of participants is performed as outlined in the scheme depicted in Fig.20

[0237] Use of co-intervention

[0238] Investigational product

[0239] The investigational products of the current study will be EAA powder mix (PYC Laboratoire), NEAA powder mix (PYC Laboratoire), and maltodextrin powder (AMACX). A non-caloric sweetener powder (Stevia, Canderel, Sligro) will be added to all beverages after the powders are weighed into appropriate tubes of specific doses for each participant. Throughout the 10- days, subjects will receive beverages containing EAA+NEAA, just EAA, or EAA+maltodextrin powder four times a day. The amount will be based on body weight, height, experimental group, and energy requirements. All beverages will be provided to participants in the appropriate powder form within sterile 75 mL containers to pour into and mix in shaker bottles. Participants will be instructed to fill the bottles to a certain fill line (300 mL) and mix prior to consumption. The amino acid powders will be manufactured by PYC Laboratoire (using a similar composition as previously used by our group in an METC-approved published study (20)). The maltodextrin powder will be from AMACX . The non-caloric sweetener will be Stevia (Canderel, from Sligro,. The powders will be stored in a sealed container at room temperature in a dry and locked location. Powders will be prepared for participants according to Standard Operating Procedure and given to participants in tubes to mix in shaker bottles. Participants will be provided with a log sheet and labels explaining the preparation of beverages from the powder. Participants will also be provided additional (liquid) flavouring to add to the beverages when mixed with water. The additional flavouring will be FlavDrops (My Protein). Participants will be instructed to add 10 drops of flavouring to the beverage prior to drinking as indicated in the meal menu overview and the labels for the protein supplement. The randomization procedure to allocate treatment group will occur via a random number generator using stratified block randomization.

[0240] INVESTIGATIONAL PRODUCT

[0241] Name and description of investigational product(s)

[0242] Participants will consume between 100 to 600, preferably 300 mL beverages containing either NEAA+EAA, EAA alone, or EAA+maltodextrin drink powder. The EAA and NEAA powders are manufactured from PYC Laboratoire (France). The maltodextrin powder is produced by AMACX . All beverages will be prepared from powders. A non-caloric sweetener powder (Stevia, Canderel will be added to all mixtures given to participants in 75 mL containers to prepare with water within an electric shaker bottle (XXL Nutrition, the Netherlands). The specific amount of the powder will be based on energy requirements of the participant (calculated using body weight, height, age, physical activity levels) and the intervention group. The maltodextrin powder acts as a placebo containing carbohydrates and does not contain any protein.

[0243] Description and justification of route of administration and dosage

[0244] Previous studies indicate that ~20 g amino acids (or protein) is sufficient to optimally stimulate muscle protein synthesis rates in young men . It is recommended to consume these moderate doses of protein repeatedly throughout the day (i.e. at 4 intervals) to maximize net protein balance. Based on body weight, height, age, and intervention group, participants will consume -22.5-40 g of powder mixture four times a day. This amount of amino acids at these intervals (-10-40 g) is sufficient for healthy intake. The amino acids will be ingested by consuming a 300 mL beverage orally, four times per day. The volume of the beverage is comparable to drinking a glass of water and has been used in previous METC-approved studies from our research group. The amount of amino acids to be consumed for each participant will be calculated as follows:

[0245] First, the revised Harris-Benedict equation will be used to calculate energy expenditure per day, with a physical activity factor of 1 .5-1.7 (depending on physical activities assessed on the screening visit using an IPAQ questionnaire):

[0246] Caloric intake per day = Physical activity facto (4.799 x body height (cm)) - (5.677 x age (years)

[0247] Based on this number, participants will then be grouped into one of nine caloric brackets, with the amounts of AA mixture for each beverage as listed below:

[0248] 1. 1750 - 2000 kcal / day: All groups 20.5g total powder mixture. Composed of: a. 7%EAA+10% NEAA group: 8.5g EAA + 12g NEAA b. 7%EAA group: 8.5g EAA + 12g maltodextrin powder c. 17%EAA group: 20.5g EAA

[0249] 2. 2000 - 2250 kcal / day: All groups 23g total powder mixture. Composed of: a. 7%EAA+10% NEAA group: 9.5g EAA + 13.5g NEAA b. 7%EAA group: 9.5g EAA + 13.5g maltodextrin powder c. 17%EAA group: 23g EAA 3. 2250 - 2500 kcal / day: All groups 25.5g total powder mixture. Composed of: a. 7%EAA+10% NEAA group: 10.5g EAA + 15g NEAA b. 7%EAA group: 10.5g EAA + 15g maltodextrin powder c. 17%EAA group: 25.5g EAA

[0250] 4. 2500 - 2750 kcal / day: All groups 28g total powder mixture. Composed of: a. 7%EAA+10% NEAA group: 11.5g EAA + 16.5g NEAA b. 7%EAA group: 11.5g EAA + 16.5g maltodextrin powder c. 17%EAA group: 28g EAA

[0251] 5. 2750 - 3000 kcal / day: All groups 30.5g total powder mixture. Composed of: a. 7%EAA+10% NEAA group: 12.5g EAA + 18g NEAA b. 7%EAA group: 12.5g EAA + 18g maltodextrin powder c. 17%EAA group: 30.5g EAA

[0252] 6. 3000 - 3250 kcal / day: All groups 33g total powder mixture. Composed of: a. 7%EAA+10%NEAA group: 13.5g EAA + 19.5g NEAA b. 7%EAA group: 13.5g EAA + 19.5g maltodextrin powder c. 17%EAA group: 33g EAA

[0253] 7. 3250 - 3500 kcal / day: All groups 33g total powder mixture. Composed of: a. 7%EAA+10% NEAA group: 13.5g EAA + 19.5g NEAA b. 7%EAA group: 13.5g EAA + 19.5g maltodextrin powder c. 17%EAA group: 33g EAA

[0254] 8. 3500 - 3750 kcal / day: All groups 39g total powder mixture. Composed of: a. 7%EAA+10%NEAA group: 16g EAA + 23g NEAA b. 7%EAA group: 16g EAA + 23g maltodextrin powder c. 17%EAA group: 39g EAA

[0255] 9. 3750 - 4000 kcal / day: All groups 41 g total powder mixture. Composed of: a. 7%EAA+10%NEAA group: 17g EAA + 24g NEAA b. 7%EAA group: 17g EAA + 24g maltodextrin powder c. 17%EAA group: 41 g EAA

[0256] Each ingredient will be weighed (EAA powder, NEAA powder, maltodextrin powder) into each container for each dose for the individual participant.

[0257] Participants will be given Flavor Drops (My Protein) in which they will be instructed to add 10 drops to each beverage prior to ingestion.

[0258] Dosages, dosage modifications and method of administration Subjects will receive their drinks in a blinded manner. Beverages will be consumed orally between 2 and 6 times a day, preferably 4 times a day for between 5 and 15 days, preferably 10 days.

[0259] Preparation and labelling of Investigational Medicinal Product The appropriate amount of powder for each beverage will be weighed and placed in sterile containers. Participants will be given an electric shaker bottle (XXL Nutrition, the Netherlands) and instructions to empty the contents of the container into the shaker bottle and mix appropriately at the time of ingestion. A “fill line” will be marked on the shaker bottle for the participant to fill to and shake to mix prior to ingestion

[0260] Study parameters / endpoints

[0261] Main study parameter / endpoint

[0262] Skeletal muscle protein synthesis rates, expressed as fractional synthesis rates (FSR, % / d), will be measured for 10 days over the dietary intervention. To calculate FSR, the standard precursor-product method will be used (14). Skeletal muscle protein synthesis rates will be calculated from:

[0263] - Saliva2H enrichment (i.e., precursor)

[0264] - Tissue protein-bound enrichment2H-alanine

[0265] The FSR will be calculated using the following formula:

[0266] FSR (% / d) = (Etissue I Eprecursor x time) * 100

[0267] Where Etissue the protein-bound2H-alanine enrichments in muscle tissues, Eprecursor is the weighted mean of the saliva2H enrichment corrected by a factor of 3.7 (to represent free plasma 2H-alanine enrichment) and time is the tracer incorporation time.

[0268] Secondary study parameters / endpoints

[0269] Secondary endpoints are muscle mass, organ volumes, resting energy expenditure, blood pressure and heart rate, plasma profiles (lipids, insulin, amino acids, inflammatory markers), mitochondrial respiration, and biochemical signaling pathways in skeletal muscle

[0270] The changes in muscle mass and organ volumes (MRI) from baseline and between the dietary intervention groups will be examined. Skin protein fractional synthesis rates (FSR) will be determined (as explained above regarding muscle FSR) using saliva2H enrichment and skin tissue protein-bound 2H enrichment. In addition, resting energy expenditure (indirect calorimetry), blood pressure and heart rate, plasma profiles (lipids, insulin, amino acids, inflammatory markers in blood), urine profiles (markers of protein breakdown), mitochondrial respiration (skeletal muscle biopsies), and biochemical signaling pathways (skeletal muscle biopsies) will be measured.

[0271] Other study parameter(s)

[0272] Other study parameters include baseline age (y), body mass (kg), height (m), physical activity levels, and habitual dietary intake. EXPERIMENT 6

[0273] Sex dependent response to dietary modulation

[0274] The apparent loss of fat mass assessed by CT was supported by reduced adipose tissue mass and adipocyte cross-sectional area of a visceral (eWAT) depot (), as well as a loss of liver lipids (see i.a. Fig. 1 Q-W). These responses did not appear to be sex dependent, as female mice similarly lost weight and visceral fat mass (Fig. 21A, B). Additionally, the key findings of weight and fat mass loss were retained in mice housed at thermoneutrality following 4 weeks of acclimation suggesting a thermogenic stress / UCP-1 independent mechanism (Fig. 21 C, D).

[0275] Additional whole -body data in both sexes was generated (Fig. 22)

[0276] EXPERIMENT 7

[0277] Lipids are mobilized to support amino acids biosynthesis

[0278] To gain insight into the possibility of the metabolic fate of the liberated lipids, metabolomics in the serum of these animals was performed. In further support of the absence of lipotoxicity in mice fed a NEAA- depleted diet, serum FFA levels were reduced by as much as -90%, and serum TAG levels were reduced by nearly -50% following consumption of the 18% diet (Fig. 24A). Various stress responsive cytokines, such as fibroblast growth factor 21 (FGF21 ) and growth differentiation factor 15 (GDF15), have been linked to obesity (Dushay et al., 2010; Vila et al., 2011 ). Indeed, we have previously shown a role for pharmacological administration of GDF15 in regulating energy expenditure (Wang et al., 2023), while others have suggested FGF21 is required for regulating weight loss and extending life with protein restricted diets (Hill et al., 2022). While GDF15 was not altered with NEAA restriction, FGF21 was increased following consumption of both NEAA depleted diets (Fig. 24B). However, FGF21 did not increase further in the 18% diet despite greater weight loss. Combined, these data suggest that changes in neither serum GDF15 nor FGF21 levels can explain the greater weight loss observed with the 18% diet (Fig. 24B). Untargeted metabolomics were applied within the serum of these mice to determine the impact of dietary interventions on circulating amino acid profiles. Serum analysis detected 721 metabolites; however, only 7 metabolites displayed an absolute Iog2 fold-change >1 (5 down, 2 up: Figure 24C). Given the small but consistent changes in serum amino acids, various amino acid metabolic pathways were identified as the most affected by our interventions (Figure 24D). However, despite the complete absence of NEAA within the diet, not all serum NEAA were reduced (Fig. 25). Additionally, while there were dramatic phenotypic differences between the two amino acid diets, virtually no NEAA was differentially affected in mice consuming the 7% and 18% diets (Fig. 25). Displaying the serum amino acid data as an integrated pathway highlights the integration of metabolic byproducts (e.g. pyruvate, oxaloacetate, D-ketoglutarate) involved in NEAA biosynthesis (Fig. 24E). In summary, the metabolomics data indicate fat mass loss was associated with the maintenance of circulating amino acids profiles following diets devoid of NEAA.

[0279] EXPERIMENT 8

[0280] To further investigate the regulatory signals underlying gene expression changes induced by the dietary intervention according to the present invention, a motif enrichment analysis was performed (Fig.26) in the promoter regions of downregulated genes using JASPAR database for transcription factors. In eWAT, motifs associated with Kruppel-like factor-5 (KLF5), Specificity protein 9 (SP9), and KLF12 were significantly enriched among the downregulated genes (Fig. 26), while examination of the liver dataset identified enrichment of motifs for KLF5 and KLF14 (Fig. 26), indicating a partially overlapping, yet tissue-specific, transcriptional regulation signature.

[0281] EXPERIMENT 9

[0282] To further validate the observed reduction in genes associated with fatty acid synthesis, and to broadly interrogate the metabolic consequences of our dietary intervention, unlabeled bottom-up proteomics was performed in liver and adipose tissue at the end of the 14-day dietary intervention. Quantitative proteomics detected -3,500 proteins in total, which were filtered to include only those consistently detected across all biological replicates within each tissue, yielding 884 high-confidence proteins in eWAT and 1 ,050 in liver tissue for differential abundance analysis. 78 proteins were found which were differentially expressed in epidydimal adipose tissue following the 2-week intervention (Fig. 27A). In the eWAT, the top 50 affected proteins included the reduction of 2 antioxidants (SOD and TKLT2), while the remaining 48 were increased and implicated primarily in branched chain and amino acid catabolism, including MMSA, ECHM, 2HIDH, IVD and ODB2 (Fig. 27B). While proteins involved in lipid synthesis were not detected, proteins involved in fat oxidation, including ACADV, ECHM, IVD and CACP were increased, supporting the interpretation that lipid storage within eWAT was reduced. Supporting this, GO enrichment analysis highlighted key processes involved in branch-chain, amino acid, cholesterol, carboxylic acid, and fatty acid catabolic processes (Fig. 27C). In further support of a global reduction in peripheral lipid storage, within the liver while only 9 individual proteins reached statistical significance (Fig. 27D), the top 50 affected proteins included strong trends for reduced FASN (p=0.058) and ACLY (p=0.108; Fig. 27E). Additionally, GO analysis highlighted cholesterol transport, fatty acid metabolic processes and fatty acid (acyl-CoA) biosynthesis within the most differentially affected pathways (Fig. 27F). Combined, the multi-omic profiling of adipose and liver tissue supports the phenotype of decreased lipid storage (decreased adipose mass and liver lipids) and suggests increased amino acid catabolism and decreased fatty acid synthesis within the adipose tissue could contribute to maintain NEAA directly (increased amino acid catabolism, particularly of BCAA’s) or possibly indirectly through provision of fatty acids as a substrate for NEAA biosynthesis.

[0283] EXPERIMENT 10

[0284] Dark and light cycling was performed to test the effect of the NEAA devoid diet according to the invention. The NEAA devoid diet also re-established metabolic flexibility, as highlighted by oscillations in wholebody VO2, RER, and carbohydrate oxidation throughout the day (Fig. 28A and Fig. 28B-E) without affecting mitochondrial respiration within skeletal muscle.

Claims

1. 47CLAIMS1 . A composition comprising a mixture of amino acids for use in the treatment of an obese or overweight subject, wherein the mixture of amino acids comprises essential amino acids and does not comprise non-essential amino acids and wherein the composition is administered orally.

2. The composition for use in accordance with claim 1 , wherein the mixture of essential amino acids administered per day per human subject, represents about 5-30, or about 15-19, or about 17% of the total caloric intake.

3. The composition for use in accordance with claim 1 or claim 2, wherein the treatment comprises furthermore a diet prescribed to the subjects based on their individual needs.

4. The composition in accordance with any of claims 1 -3, wherein the subject is a human.

5. The composition for use in accordance with claim 4, wherein the prescribed diet is a low protein diet.

6. The composition according to claim 5, wherein the low protein diet comprises protein in an amount representing at most 5%, preferably at most 4%, more preferably at most 3%, of the total caloric daily intake.

7. The composition for use in accordance with any of claim 5 or claim 6, wherein the low protein diet comprises meals, wherein with each meal a portion of the composition is concomitantly administered.

8. The composition for use in accordance with any of claims 1 -7, wherein the essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, preferably wherein the composition comprises, in percent per weight relative to the weight total of amino acids in the composition, histidine 4-8 %, isoleucine 9-13 %, leucine 18-22 %, lysine 19-23 %, methionine 4-8 %, phenylalanine 8-12 %, threonine 8-12 %, tryptophan 1-5 %, and valine 12-16 %.

9. The composition according to any of claims 1 -8, wherein in the treatment, subjects have a reduction in body weight.

10. The composition according to any of claims 1 -9, wherein in the treatment, subjects muscle mass is substantially maintained.

11. The composition according to any of claims 1 -10, wherein in the treatment, subjects have a reduction in adiposity, preferably a reduction in (epidymal) white adipose tissue and / or wherein subjects have a reduction in whole body fat.

12. The composition according to any of claims 1 -11 , wherein in the treatment, subjects have a reduced hepatic adiposity or reduced hepatic lipid accumulation.

13. The composition according to any of claims 1 -12, wherein the subjects have a diabetic cardiomyopathy, and wherein in the treatment, diabetic cardiomyopathy is reversed.

14. The composition according to any of claims 1 -13, wherein in the treatment, cardiometabolic health is improved, comprising one or more of reduced fasting blood glucose levels, reducing serum lipids, improving glucose tolerance, improving cardiac output .

15. The composition according to any of claims 1 -14, wherein the subject is not an overweight or obese subject.

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